Waveguide-based optical scanning systems

ABSTRACT

A scanning sensor system, methods of use and kits for detecting a biologically active analyte are provided. The scanning senor system includes a light source, a detector, a substrate comprising a plurality of waveguides and a plurality of optical sensing sites in optical communication with one or more waveguide of the substrate, and at least one adapter configured to couple with the substrate and provide optical communication between the light source, the waveguides of the substrate, and the detector.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 12/209,295, filed Sep. 12, 2008, titled “Waveguide-BasedOptical Scanning Systems,” now Publication No. US-2009-0068668-A1, whichclaims the benefit of U.S. Provisional Application No. 60/971,878, filedSep. 12, 2007, which applications are incorporated herein by reference.This application is also a continuation-in-part of U.S. patentapplication Ser. No. 13/474,130, filed May 17, 2012, titled “OpticalScanning System,” now Publication No. US-2012-0231532-A1, which is acontinuation of U.S. patent application Ser. No. 13/109,280, filed May17, 2011, titled “Optical Scanning System,” now U.S. Pat. No. 8,187,866,which is a continuation application of U.S. patent application Ser. No.11/683,808, filed Mar. 8, 2007, titled “Optical Scanning System,” nowU.S. Pat. No. 7,951,583, which application claims the benefit of U.S.Provisional Application No. 60/743,458, filed Mar. 10, 2006, entitled“Optical Scanning System.”

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BACKGROUND

Biological substance analysis methods based on optical means have risenin popularity in the last couple of decades. Common to all these methodsis that chemical interactions between the bio-molecules produce changesthat affect some measurable optical properties, such as emissionspectrum, absorption spectrum and index of refraction. The changes inthe optical properties can occur in the analyte itself or through amediator such as the surface on which the interaction takes place. Thesechanges are then monitored using a beam of incoming light (usually laserlight) which in-turn changes the outgoing light spectrum (e.g. influorescence), intensity (e.g. in absorption), or phase (e.g. SurfacePlasmon Resonance=SPR and any kind of interferometric method).

While most of these optical bio-analysis methods have found nicheapplications and markets, one method that became highly popular andinfluential was microarray optical fluorescence scanning. Such opticalscanning has enabled running tests on tens of thousands of miniaturesamples in a relatively short period of time. The major advantages ofthis method include: a) performance (sensitivity and Signal to NoiseRatio=SNR); b) speed; and c) miniaturization of the sampled analyte.These parameters define the efficiency and superiority of the method.

Currently microarray elements are spotted on top of a flat substratechip usually made of glass, plastic or epoxy. Subsequently, the chip isscanned using confocal scanning systems where the exciting light and theresulting fluorescence light are both shined and collected from aboveand analyzed using a single photo-multiplier (PMT) detector. Thisarrangement suffers from several inherent limitations including a veryshort interaction length between the bio -sample and the light (usuallya single mono-layer). This limits the signal strength and thus the SNR.Another limitation is a high background or noise due to the fact thatthe back reflected light and the emitted fluorescent light travel in thesame direction. A further limitation is high sensitivity to theplanarity and the position of the chip that need to be maintained infocus. Still another limitation is slow operation due to the need tohave large enough number of ‘pixels’ (scanned spots) within every sampleand long enough integration time. Yet another limitation is the need fora complicated optical and mechanical structure that entails bulky andexpensive systems.

Another optical bio-analysis method is waveguide based bio-sensors.Bio-sensing based on waveguides has been around for a while. Thesebiosensors can be divided into three main categories. The first involveslab waveguide fluorescence excitation with light collection from aboveor below the chip. In this arrangement the bio-analyzed spots arelocated on the surface of a chip that contains a single slab-waveguide.Light is coupled into the waveguide using a lens or a grating thatexcites the entire chip with all its bio-analyzed spots simultaneously.The fluorescence is collected using an optical imaging system and aCharge-Coupled Device (CCD) detector from above or underneath the chip.One drawback of this kind of systems is relatively poor performance dueto uniformity of excitation as well as collection of the light. Thisleads to non-repeatable results. Another drawback is high noise levelsdue to crosstalk between the different spots. A further drawback is thatlarge spots and relatively small numbers of elements are required togenerate signal large enough for efficient imaging with the CCD. Yetanother drawback is the long integration time to overcome SNR issues.Examples of the above method are described in U.S. Pat. Nos. 5,814,565;6,911,344 and 6,395,558.

A second waveguide based bio-sensor utilizes an interferometric opticaldevice. In this case, channel waveguides are used together withinterferometric devices such as Mach Zehender Interferometers (MZI) orring-resonators. These sensitive interferometric devices sense thechange in the index of refraction due to binding of the bio-moleculesnear a waveguide surface. The major problems associated with this typeof systems include non-specificity due to inability to recognize theexact reason for the index change which may occur from other materialdeposition as well as temperature changes. Another problem is a veryslow speed in addressing the different elements which disqualify thismethod for running large numbers of element arrays. Examples of theabove method are described in U.S. Pat. Nos. 5,494,798 4,515,430,5,623,561 and 6,618,536.

A third waveguide based bio-sensor utilizes Surface Plasmon Resonance(SPR). Here, in one example, a thin gold layer deposited on top of aglass substrate. The bio-analyzed sample on top of the gold induceschanges in the refractive index above the gold layer and thus changingthe resonant angle for generating surface Plasmons along the gold layer.The Plasmons generation is detected as an enhanced peak in the reflectedbeam. Examples of the SPR method are covered, for example, in U.S. Pat.No. 6,956,651 B2. Other types of optical bio -sensors and array scannersexist such as described in U.S. Pat. No. 6,396,995 B1.

SUMMARY OF THE DISCLOSURE

In general, in one aspect, the invention features a scanning sensorsystem, methods and kits for use thereof including a light source, adetector, a substrate and a plurality of optical sensing sites. Thesubstrate is in optical communication with the light source and thedetector either directly or indirectly, for example, through an adapterchip. Additionally, the substrate includes a plurality of substantiallyparallel waveguides used both for guiding the excitation light to thesensing sites and for collecting the emitted light from the sensingsites. The plurality of optical sensing sites can each be in opticalcommunication with a waveguide.

Implementations of the invention can include one or more of thefollowing features.

In general in one aspect a scanning sensor system for detecting abiologically active analyte is provided including a light source, adetector, a substrate comprising a plurality of waveguides and aplurality of optical sensing sites in optical communication with one ormore waveguide of the substrate, and at least one adapter configured tocouple with the substrate and provide optical communication between thelight source, the waveguides of the substrate, and the detector.

The substrate and the at least one adapter in some embodiments can besubstantially planar and make up or comprise a planar lightwave circuit.The at least one adapter can be further configured to removably couplewith the substrate. The at least one adapter can be further coupled toat least one of the light source and the detector. The at least oneadapter can in one embodiment be a single adapter further coupled to thelight source and the detector.

Coupling of the system components can be by fiber optic.

The adapter in some embodiments includes a plurality of edges andcoupling of the adapter to the light source and the detector includescoupling to a first edge of the adapter and coupling to the substratecomprises coupling at a second edge of the adapter.

The plurality of waveguides of the substrate and/or the adapter caninclude in-coupling waveguides and out-coupling waveguides. Thein-coupling waveguides can be coupled to the out-coupling waveguidesthrough a combiner. In one embodiment the substrate further includes atleast one combiner. In another embodiment the adapter further includesat least one combiner.

In a particular embodiment the at least one adapter is a single adapter,the plurality of waveguides include in-coupling waveguides andout-coupling waveguides, wherein the in-coupling waveguides are coupledto the out-coupling waveguides through a combiner, wherein the lightsource comprises a light generator element coupled to at least onein-coupling waveguide, and wherein the detector includes a detectorelement coupled to at least one out -coupling waveguide.

The optical sensing site can include a sensor configured to transduce afirst light wave generated by the light source in a waveguide, resultingin a second light wave in the same waveguide, the second light wavebeing detectable by the detector.

The plurality of waveguides can be in-coupling waveguides coupled toout-coupling waveguides by way of combiners, and a first light wave canbe carried by an in-coupling waveguide and a second light wave can becarried by an out-coupling waveguide. In one embodiment the adapter andsubstrate include inter-coupling optically communicating in-couplingwaveguides and out-coupling waveguides and the substrate furtherincludes the combiners.

The sensor can include a biologically active analyte in a sample, andwherein a measurable change in a first light wave results when thesensor discriminates or interacts with the biologically active analyte.In one emobidment the sensor is adapted to support an immunoassay. Theimmunoassay supported can be an enzyme-linked immunosorbent assay(ELISA). The immunoassay supported can be a fluorescent immunoassay.

The sensor can be selected from the group including a fluorescence well,an absorption cell, an interferometric sensor, a diffractive sensor andsurface plasmon resonance sensor.

The biologically active analyte can be selected from the group includinga nucleic acid, a protein, an antigen, an antibody, a microorganism, agas, a chemical agent and a pollutant. In one embodiment the nucleicacid is produced via an amplification reaction.

The waveguides can be single-mode. The waveguides can alternatively bemulti-mode. In one embodiment the waveguides are single-mode in thevertical dimension and multi-mode in the lateral dimension.

The optical sensing sites can include wells. In one embodiment theoptical sensing sites include the surface of the substrate above thewaveguides. In another embodiment the optical sensing sites includebiochemical interaction sites. In a further embodiment the opticalsensing sites include optical transducers. The optical transducers caninlcude fluorescence wells comprising fluorescent or luminescentcompounds, wherein light waves guided by a waveguide of the plurality ofwaveguides excite the fluorescent or luminescent compound in the wellsin the presence of a biologically active analyte, and the same waveguidecollects and guides light emitted from the wells to the detector.

The number of optical sensing sites can be greater than 10, greater than200, or greater than 5,000.

The light source can be switchable or passive. In one embodiment thelight source includes a dynamic light source. In one embodiment thelight source is a passive 1×N splitter with N being between 1 and 1000.

The light source can include a chip containing an array of lightgenerators coupled to an array of waveguides. In one embodiment thelight source is an optical switch including a light generator coupled toone or more input of the optical switch. In another embodiment theoptical switch further includes a branched architecture. The opticalswitch can further include one or more inputs and multiple outputs. Theoptical switch can further include greater than about 10 outputs,greater than about 100 outputs or greater than about 1,000 outputs. Inone embodiment the optical switch further includes substantially between20 and 200 outputs.

The light generator can provide variable wavelengths of light. The lightgenerator can be selected from the group including a broad-band source,a source with one or more discrete spectral lines and a tunable source.

The light source can be butt-coupled to one or more of the at least oneadapter. In one embodiment the light source includes one or morewaveguide and is evanescently coupled to the at least one adapterthrough a proximate arrangement of the one or more light sourcewaveguide and one or more waveguide of the at least one adapter.

The detector can be a photodetector array. In one embodiment thedetector is a plurality of detectors. In another embodiment two or moredetectors are coupled to and in optical communication with one or morewaveguide of the at least one adapter at one or more edges of the atleast one adapter.

The system can further include a thermal transfer element in thermalcommunication with the substrate. In one embodiment the thermal transferelement is a thermoelectric cooler. In another embodiment each opticalsensing site includes a thermal transfer element in thermalcommunication with the optical sensing site. The thermal transferelement can include a thin-film heater. In one embodiment each opticalsensing site further includes a thermistor in thermal communication withthe optical sensing site.

The substrate can further include one or more microchannel and one ormore reservoirs in fluid communication with one or more optical sensingsite. In one embodiment the system further includes a fluidics layercoupled to the substrate and includes one or more microchannel and oneor more reservoirs in fluid communication with one or more opticalsensing site.

In general in another aspect a scanning sensing method is providedincluding coupling a removable substrate including a plurality ofin-coupling waveguides, a plurality of out-coupling waveguides andcombiners for coupling the in-coupling and out-coupling waveguides,wherein the substrate is coupled in optical communication with anadapter, a light source, and a detector to provide a scanning sensorsystem, wherein the adapter is coupled to the light source and thedetector. The method further includes delivering a sample suspected ofcontaining a biologically active analyte to be detected to an opticalsensing site of the substrate, providing a first light wave using alight source to one or more of the plurality of in-coupling waveguidesof the substrate, wherein the in-coupling waveguides are in opticalcommunication with the optical sensing site, wherein the first lightwave is transducable by a sensor associated with the optical sensingsite to a second light wave carried to an out-coupling waveguide, anddetecting a measurable change in the second light wave using thedetector, wherein a measurable change in the first light waves occurswhen the sensor interacts with the biologically active analyte.

Scanning sensing can further include switching one or more input lightwave from the light source into the substrate to produce the first lightwave in one or more of the in-coupling waveguides. In one embodiment thelight source includes an optical switch for controlled switching of oneor more input light wave, the optical switch can multicast light to aplurality of outputs and into the substrate to controllably produce thefirst light wave in one or more of the in-coupling waveguides. The lightsource can inlcude an array of individually controlled light generatorsfor controlled switching of one or more input light wave, tocontrollably produce the first light wave in one or more of thein-coupling waveguides.

The method can further include simultaneously detecting the second lightwave with the detector wherein the detector comprises a photodetectorarray.

In one embodiment a portion of the sensing sites include referencesample material for calibration and/or normalization.

The biologically active analyte can be selected from the groupconsisting of a nucleic acid, a protein, an antigen, an antibody, amicroorganism, a gas, a chemical agent and a pollutant. In oneembodiment the biologically active analyte is a protein. In anotherembodiment a single nucleotide polymorphism (SNP) is detected in thebiologically active analyte. In a further embodiment expression of agene is detected upon detection of the biologically active analyte.

The sensor can be adapted to support an immunoassay and wherein thesensor interacting with the biologically active analyte includes anoutcome of an immunoassay. In one embodiment the immunoassay supportedis an enzyme-linked immunosorbent assay (ELISA). The immunoassaysupported can be a fluorescent immunoassay.

Detecting a measurable change in the second lightwave can provide adiagnostic result.

The method can further include conducting a real-time PCR reaction atthe optical sensing site.

In general in another aspect a kit for assaying a sample for abiologically active analyte is provided including a scanning sensorsystem including a light source, a detector, a substrate comprising aplurality of waveguides and a plurality of optical sensing sites inoptical communication with one or more waveguide of the substrate, andat least one adapter configured to couple with the substrate and provideoptical communication between the light source, the waveguides of thesubstrate, and the detector. The kit further includes packaging andinstructions for use of the system. In one embodiment the adapter andsubstrate includes a planar lightwave circuit. In another embodiment theoptical sensing sites includes a sensor adapted to support animmunoassay, and wherein the kit further includes one or moreimmunoassay reagents.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present methods and compositions may be obtained byreference to the following detailed description that sets forthillustrative embodiments, in which the principles of our methods,compositions, devices and apparatuses are utilized, and the accompanyingdrawings of which:

FIG. 1A is a schematic of the scanning sensing system according to oneembodiment of the invention including a switchable light source, fibers,an adapter chip, a substrate, optical sensing sites and a detector.

FIG. 1B is a schematic of the scanning sensing system according toanother embodiment of the invention including a switchable light source,fibers, an adapter chip, a substrate, optical sensing sites and adetector.

FIG. 1C is a schematic of the scanning sensing system according toanother embodiment of the invention a including a switchable lightsource, fibers, two adapter chips, a substrate, optical sensing sitesand a detector.

FIG. 1D is a schematic of the scanning sensing system according toanother embodiment of the invention including a switchable light sourceand detector chip, a substrate, and optical sensing sites.

FIG. 2 is block diagram showing the scanning system of the invention ina housing as part of a working system.

FIG. 3A is a schematic of the substrate of the invention according toone embodiment including optical waveguides in conjunction with opticalsensing sites and barriers.

FIG. 3B is a schematic of the substrate of the invention according toanother embodiment including optical waveguides and combiners inconjunction with optical sensing sites and barriers.

FIG. 3C is a schematic cross section of the substrate of the inventionaccording to one embodiment including an optical waveguide inconjunction with an optical sensing site.

FIG. 4A is a schematic of the adapter chip of the invention according toone embodiment including optical in-coupling and out-coupling waveguidesand the optical combiners.

FIG. 4B is a schematic of the adapter chip of the invention according tothe second embodiment including the optical in-coupling and out-couplingwaveguides.

FIG. 4C is a schematic of a side view of the substrate in relation to athermoelectric cooler.

FIG. 4D is a schematic of the substrate of the invention illustratingdetails of an optical sensing site including a heater and a thermistor.

FIG. 4E is a schematic of the substrate of the invention includingreservoirs and micro channels in relation to optical sensing sites.

FIG. 5A is a schematic of a general substrate including typical layersand waveguides representative of those of the current invention.

FIG. 5B is a photomicrograph image of waveguides representative of thoseof the invention and a silica layer.

FIG. 5C is a perspective view of waveguides and associated substratelayers.

FIG. 6A is a schematic of a switchable light source of the inventionincluding inputs and outputs.

FIG. 6B is a schematic of a branched architecture between the inputs andoutputs of a switchable light source of the invention.

FIG. 6C is a schematic of one embodiment of a switchable light sourceand detector chip of the invention including light generator elements,detector elements, in-coupling and out-coupling waveguides andcombiners.

FIG. 7 is a schematic of a detector of the invention.

FIG. 8 is a block diagram showing a representative example logic devicein communication with an apparatus for use with the scanning sensingsystem of the invention.

FIG. 9 is a block diagram showing a representative example of a kit.

FIGS. 10A-D are schematics illustrating a representative manufacturingprocess for the substrate and waveguides of the invention.

FIG. 11 is a flow chart showing a representative manufacturing processfor the substrate.

DETAILED DESCRIPTION

Apparatus, methods, and kits for optical sensing, using a scanningsensing system including a light source, a detector, at least oneadapter, a substrate and a plurality of optical sensing sites areprovided. Related scanning sensing systems, methods and kits includingsuch systems have been previously described in U.S. patent applicationSer. No. 11/683,808 filed March 8, 2007, the entire contents of whichare incorporated herein by reference. The substrate of the presentsystem includes a plurality of substantially parallel waveguides and aplurality of sensing sites. Advantageously the at least one adapter ofthe system can provide for removable coupling of the substrate withsystem components. The optical sensing sites include a sensor and are inoptical communication with one or more waveguides. Sensing of a varietyof environmental and biological samples can be achieved using theapparatus, methods and kits described herein. The general theoreticalprinciples of lightwave guiding and evanescent field fluorescenceexcitation apply to the embodiments disclosed herein.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the inventions described herein belong. Although anymethods, devices, and materials similar or equivalent to those describedherein can be used in the practice or testing of the inventionsdescribed herein, the preferred methods, devices and materials are nowdescribed.

A biologically active analyte can be any substance which can affect anyphysical or biochemical properties of a biological organism, includingbut not limited to viruses, bacteria, fungi, plants, animals, andhumans. In particular as used herein, biologically active analyteaccording to the present invention includes without limitation drugs,prodrugs, pharmaceutical agents, drug metabolites, biomarkers such asexpressed proteins and cell markers, antibodies, serum proteins,cholesterol, polysaccharides, nucleic acids, biological analytes, gene,protein, or hormone, or any combination thereof. A biologically activeanalyte can further include a natural or man-made substance includingbut not limited to a gas, a chemical agent or a pollutant, or acombination thereof (e.g., from an environmental source). At a molecularlevel, the biologically active analytes can be polypeptide glycoprotein,polysaccharide, lipid, nucleic acid, and a combination thereof.

Of particular interest are biomarkers associated with a particulardisease or with a specific disease stage.

Such biologically active analytes include but are not limited to thoseassociated with autoimmune diseases, obesity, hypertension, diabetes,neuronal and/or muscular degenerative diseases, cardiac diseases,endocrine disorders, any combinations thereof.

Also of interest are biomarkers that are present in varying abundance inone or more of the body tissues including heart, liver, prostate, lung,kidney, bone marrow, blood, skin, bladder, brain, muscles, nerves, andselected tissues that are affected by various disease, such as differenttypes of cancer (malignant or non-metastatic), autoimmune diseases,inflammatory or degenerative diseases.

Also of interest are biologically active analytes that are indicative ofa microorganism. Exemplary microorganisms include but are not limited tobacterium, virus, fungus and protozoa. Biologically active analytes thatcan be detected by the subject method also include blood-born pathogensselected from a non-limiting group that consists of Staphylococcusepidermidis, Escherichia coli, methicillin-resistant Staphylococcusaureus (MSRA),

Staphylococcus aureus, Staphylococcus hominis, Enterococcus faecalis,Pseudomonas aeruginosa, Staphylococcus capitis, Staphylococcus warneri,Klebsiella pneumoniae, Haemophilus influnzae, Staphylococcus simulans,Streptococcus pneumoniae and Candida albicans.

Biologically active analytes that can be detected by the subject deviceand methods also encompass a variety of sexually transmitted diseasesselected from the following: gonorrhea (Neisseria gorrhoeae), syphilis(Treponena pallidum), chlamydia (Chlamydia tracomitis), nongonococcalurethritis (Ureaplasma urealyticum), yeast infection

(Candida albicans), chancroid (Haemophilus ducreyi), trichomoniasis(Trichomonas vaginalis), genital herpes (HSV type I and II), HIV I, HIVII and hepatitis A, B, C, G, as well as hepatitis caused by TTV.

Additional biologically active analytes that can be detected by thesubject apparatus and methods encompass a diversity of respiratorypathogens including but not limited to Pseudomonas aeruginosa,methicillin-resistant Staphylococcus aureus (MSRA), Klebsiellapneumoniae, Haemophilis influenzae, Staphylococcus aureus,Stenotrophomonas maltophilia, Haemophilis parainfluenzae, Escherichiacoli, Enterococcus faecalis, Serratia marcescens, Haemophilisparahaemolyticus, Enterococcus cloacae, Candida albicans, Moraxiellacatarrhalis,

Streptococcus pneumoniae, Citrobacter freundii, Enterococcus faecium,Klebsiella oxytoca, Pseudomonas fluorsecens, Neisseria meningitidis,Streptococcus pyogenes, Pneumocystis carinii, Klebsiella pneumoniae,Legionella pneumophila, Mycoplasma pneumoniae, and Mycobacteriumtuberculosis.

Listed below are additional exemplary markers according to the presentinvention: Theophylline, CRP, CKMB, PSA, Myoglobin, CA125, Progesterone,TxB2, 6-keto-PGF-1-alpha, and Theophylline, Estradiol, Lutenizinghormone, High sensitivity CRP, Triglycerides, Tryptase, Low densitylipoprotein Cholesterol, High density lipoprotein Cholesterol,Cholesterol, IGFR.

Exemplary liver markers include without limitation LDH, (LD5), (ALT),Arginase 1 (liver type), Alphafetoprotein (AFP), Alkaline phosphatase,Alanine aminotransferase, Lactate dehydrogenase, and Bilirubin.

Exemplary kidney markers include without limitation TNFa Receptor,Cystatin C, Lipocalin-type urinary prostaglandin D, synthatase (LPGDS),Hepatocyte growth factor receptor, Polycystin 2, Polycystin 1,Fibrocystin, Uromodulin, Alanine, aminopeptidase,N-acetyl-B-D-glucosaminidase, Albumin, and Retinol-binding protein(RBP).

Exemplary heart markers include without limitation Troponin I (TnI),Troponin T (TnT), CK, CKMB, Myoglobin, Fatty acid binding protein(FABP), CRP, D-dimer, S-100 protein, BNP, NT-proBNP, PAPP-A,Myeloperoxidase (MPO), Glycogen phosphorylase isoenzyme BB (GPBB),Thrombin Activatable Fibrinolysis Inhibitor (TAFI), Fibrinogen, Ischemiamodified albumin (IMA), Cardiotrophin-1, and MLC-I (Myosin LightChain-I).

Exemplary pancreas markers include without limitation Amylase,Pancreatitis-Associated protein (PAP-1), and Regeneratein proteins(REG).

Exemplary muscle tissue markers include without limitation Myostatin.

Exemplary blood markers include without limitation Erythopoeitin (EPO).

Exemplary bone markers include without limitation, Cross-linkedN-telopeptides of bone type I collagen (NTx), Carboxyterminalcross-linking telopeptide of bone collagen, Lysyl-pyridinoline(deoxypyridinoline), Pyridinoline, Tartrate-resistant acid phosphatase,Procollagen type I C propeptide, Procollagen type I N propeptide,Osteocalcin (bone gla-protein), Alkaline phosphatase, Cathepsin K, COMP(Cartilage Oligomeric Matrix Protein), Osteocrin, Osteoprotegerin (OPG),RANKL, sRANK, TRAP 5 (TRACP 5), Osteoblast Specific Factor 1 (OSF-1,Pleiotrophin), Soluble cell adhesion molecules, sTfR, sCD4, sCD8, sCD44,and Osteoblast Specific Factor 2 (OSF 2, Periostin).

In some embodiments markers according to the present invention aredisease specific. Exemplary cancer markers include without limitationPSA (total prostate specific antigen), Creatinine, Prostatic acidphosphatase, PSA complexes, Prostrate-specific gene-1, CA 12-5,Carcinoembryonic Antigen (CEA), Alpha feto protein (AFP), hCG (Humanchorionic gonadotropin), Inhibin, CAA Ovarian C1824, CA 27.29, CA 15-3,CAA Breast C1924, Her-2, Pancreatic, CA 19-9, Carcinoembryonic Antigen,CAA pancreatic, Neuron-specific enolase, Angiostatin. DcR3 (Solubledecoy receptor 3), Endostatin, Ep-CAM (MK-1), Free Immunoglobulin LightChain Kappa, Free Immunoglobulin Light Chain Lambda, Herstatin,Chromogranin A, Adrenomedullin, Integrin, Epidermal growth factorreceptor, Epidermal growth factor receptor-Tyrosine kinase,Pro-adrenomedullin N-terminal 20 peptide, Vascular endothelial growthfactor, Vascular endothelial growth factor receptor, Stem cell factorreceptor, c kit/KDR, KDR, and Midkine.

Exemplary infectious disease markers include without limitation Viremia,Bacteremia, Sepsis, PMN Elastase, PMN elastase/α1-PI complex, SurfactantProtein D (SP-D), HBVc antigen, HBVs antigen, Anti-HBVc, Anti-HIV,Tsuppressor cell antigen, T-cell antigen ratio, T-helper cell antigen,Anti-HCV, Pyrogens, p24 antigen, Muramyldipeptide.

Exemplary diabetes markers include without limitation C-Peptide,Hemoglobin A1c, Glycated albumin, Advanced glycosylation end products(AGEs), 1,5-anhydroglucitol, Gastric Inhibitory Polypeptide, Glucose,Hemoglobin, ANGPTL3 and 4.

Exemplary inflammation markers include without limitation Rheumatoidfactor (RF), Antinuclear Antibody (ANA), C-reactive protein (CRP), ClaraCell Protein (Uteroglobin).

Exemplary allergy markers include without limitation Total IgE andSpecific IgE.

Exemplary autism markers include without limitation Ceruloplasmin,Metalothioneine, Zinc, Copper, B6, B12, Glutathione, Alkalinephosphatase, and Activation of apo -alkaline phosphatase.

Exemplary coagulation disorders markers include without limitationb-Thromboglobulin, Platelet factor 4, Von Willebrand factor.

In some embodiments a marker may be therapy specific. COX inhibitorsinclude without limitation TxB2 (Cox-1), 6-keto-PGF-1-alpha (Cox 2),11-Dehydro-TxB-1a (Cox-1).

Other markers of the present include without limitation Leptin, Leptinreceptor, and Procalcitonin, Brain S100 protein, Substance P,8-Iso-PGF-2a.

Exemplary geriatric markers include without limitation, Neuron-specificenolase, GFAP, and S100B.

Exemplary markers of nutritional status include without limitationPrealbumin, Albumin, Retinol-binding protein (RBP), Transferrin,Acylation-Stimulating Protein (ASP), Adiponectin, Agouti-Related Protein(AgRP), Angiopoietin-like Protein 4 (ANGPTL4, FIAF), C-peptide, AFABP(Adipocyte Fatty Acid Binding Protein, FABP4), Acylation-StimulatingProtein (ASP), EFABP (Epidermal Fatty Acid Binding Protein, FABP5),Glicentin, Glucagon, Glucagon-Like Peptide-1, Glucagon-Like Peptide-2,Ghrelin, Insulin, Leptin, Leptin Receptor, PYY, RELMs, Resistin, andsTfR (soluble Transferrin Receptor).

Exemplary markers of Lipid metabolism include without limitationApo-lipoproteins (several), Apo-A1, Apo-B, Apo-C-CII, Apo-D, Apo-E.

Exemplary coagulation status markers include without limitation FactorI: Fibrinogen, Factor II: Prothrombin, Factor III: Tissue factor, FactorIV: Calcium, Factor V: Proaccelerin, Factor VI, Factor VII:Proconvertin, Factor VIII:, Anti-hemolytic factor, Factor IX: Christmasfactor, Factor X: Stuart-Prower factor, Factor XI: Plasma thromboplastinantecedent, Factor XII: Hageman factor, Factor XIII: Fibrin-stabilizingfactor, Prekallikrein, High molecular-weight kininogen, Protein C,Protein S, D-dimer, Tissue plasminogen activator, Plasminogen, a2Antiplasmin, Plasminogen activator inhibitor 1 (PAI1).

Exemplary monoclonal antibody markers include those for EGFR, ErbB2, andIGF1R.

Exemplary tyrosine kinase inhibitor markers include without limitationAb1, Kit, PDGFR, Src, ErbB2, ErbB 4, EGFR, EphB, VEGFR1-4, PDGFRb, FLt3,FGFR, PKC, Met, Tie2, RAF, and TrkA.

Exemplary Serine/Threonine Kinase Inhibitor markers include withoutlimitation AKT, Aurora A/B/B, CDK, CDK (pan), CDK1-2, VEGFR2, PDGFRb,CDK4/6, MEK1-2, mTOR, and PKC-beta.

GPCR target markers include without limitation Histamine Receptors,Serotonin Receptors, Angiotensin Receptors, Adrenoreceptors, MuscarinicAcetylcholine Receptors, GnRH Receptors, Dopamine Receptors,Prostaglandin Receptors, and ADP Receptors.

A therapeutic agent can be any substances that have therapeutic utilityand/or potential. Such substances include but are not limited tobiological or chemical compounds such as a simple or complex organic orinorganic molecules, peptides, proteins (e.g. antibodies) or apolynucleotides (e.g. anti-sense). Other therapeutic agents include avast array of compounds that can be synthesized, for example, polymers,such as polypeptides and polynucleotides, and synthetic organiccompounds based on various core structures. In addition, various naturalsources can provide compounds for screening, such as plant or animalextracts, and the like. It should be understood, although not alwaysexplicitly stated that the agent is used alone or in combination withanother agent, having the same or different biological activity as theagents identified by the inventive screen. The agents and methods alsoare intended to be combined with other therapies.

Pharmacodynamic (PD) parameters according to the present inventioninclude without limitation physical parameters such as temperature,heart rate/pulse, blood pressure, and respiratory rate, and biomarkerssuch as proteins, cells, and cell markers. Biomarkers could beindicative of disease or could be a result of the action of a drug.Pharmacokinetic (PK) parameters according to the present inventioninclude without limitation drug and drug metabolite concentration.Identifying and quantifying the PK parameters rapidly from a samplevolume is extremely desirable for proper safety and efficacy of drugs.If the drug and metabolite concentrations are outside a desired rangeand/or unexpected metabolites are generated due to an unexpectedreaction to the drug, immediate action may be necessary to ensure thesafety of the patient. Similarly, if any of the PD parameters falloutside the desired range during a treatment regime, immediate actionmay have to be taken as well.

In preferred embodiments physical parameter data is stored in orcompared to stored profiles of physical parameter data in abioinformatics system which may be on an external device incorporatingpharmacogenomic and pharmacokinetic data into its models for thedetermination of toxicity and dosing. Not only does this generate datafor clinical trials years prior to current processes but also enablesthe elimination of current disparities between apparent efficacy andactual toxicity of drugs through real-time continuous monitoring. Duringthe go/no go decision process in clinical studies, large scalecomparative population studies can be conducted with the data stored onthe database. This compilation of data and real-time monitoring allowsmore patients to enter clinical trials in a safe fashion earlier thancurrently allowed. In another embodiment biomarkers discovered in humantissue studies can be targeted by the scanning sensing system forimproved accuracy in determining drug pathways and efficacy in cancerstudies.

A nucleic acid can be deoxyribonucleotides, deoxyribonucleosides,ribonucleosides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. Nucleic acids may contain knownanalogues of natural nucleotides which have similar binding propertiesas the reference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Nucleic acids may also includeoligonucleotide analogs including PNA (peptidonucleic acid), analogs ofDNA used in antisense technology (phosphorothioates, phosphoroamidates,and the like). Unless otherwise indicated, a particular nucleic acidsequence also implicitly encompasses conservatively modified variantsthereof (including but not limited to, degenerate codon substitutions)and complementary sequences as well as the sequence explicitlyindicated. Specifically, degenerate codon substitutions may be achievedby generating sequences in which the third position of one or moreselected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)).

Microorganism can include but is not limited to bacteria,actinomycetales, cyanobacteria (unicellular algae), fungi, protozoa,animal cells or plant cells or virus. Examples of microorganisms includebut are not limited to pathogens.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-natural amino acid. As used herein, the terms encompass amino acidchains of any length, including full length proteins (i.e., antigens),wherein the amino acid residues are linked by covalent peptide bonds. Inaddition, proteins that contain multiple polypeptide chains thatassociate through covalent and/or non-covalent interactions are alsoencompassed by “protein,” as used herein.

A polymorphism is the occurrence of two or more genetically determinedalternative sequences or alleles in a population. A polymorphic markeror site is the locus at which divergence occurs. Preferred markers haveat least two alleles, each occurring at frequency of greater than 1%,and more preferably greater than 10% or 20% of a selected population. Apolymorphism may comprise one or more base changes, an insertion, arepeat, or a deletion. A polymorphic locus may be as small as one basepair. Polymorphic markers include restriction fragment lengthpolymorphisms, variable number of tandem repeats (VNTR's), hypervariableregions, minisatellites, dinucleotide repeats, trinucleotide repeats,tetranucleotide repeats, simple sequence repeats, and insertion elementssuch as Alu. The first identified allelic form is arbitrarily designatedas the reference form and other allelic forms are designated asalternative or variant alleles. The allelic form occurring mostfrequently in a selected population is sometimes referred to as thewildtype form. Diploid organisms may be homozygous or heterozygous forallelic forms. A diallelic polymorphism has two forms. A triallelicpolymorphism has three forms.

A single nucleotide polymorphism (SNP) occurs at a polymorphic siteoccupied by a single nucleotide, which is the site of variation betweenallelic sequences. The site is usually preceded by and followed byhighly conserved sequences of the allele (e.g., sequences that vary inless than 1/100 or 1/1000 members of the populations).

A single nucleotide polymorphism usually arises due to substitution ofone nucleotide for another at the polymorphic site. A transition is thereplacement of one purine by another purine or one pyrimidine by anotherpyrimidine. A transversion is the replacement of a purine by apyrimidine or vice versa. Single nucleotide polymorphisms can also arisefrom a deletion of a nucleotide or an insertion of a nucleotide relativeto a reference allele.

An individual includes but is not limited to a human being, but may alsoinclude other organisms including but not limited to mammals, plants,bacteria or cells derived from any of the above.

Aspects of the invention may include one or more of the followingadvantageous features. Dense and accurate integration of opticalmanipulating elements can be achieved using planar lightwave circuitstechnology. Applications for planar lightwave circuits as describedherein include new drug discovery and development, disease research,biomarkers discovery, SNP association studies including toxicology anddisease susceptibility, and diagnostics including identifying patientspredisposed to diseases and identifying patients with particular drugsensitivity.

FIG. 1A illustrates an exemplary scanning sensing system 100 of theinvention including a switchable light source 102, an adapter chip 114,a substrate 104, optical sensing sites 112 and a detector 106. Althougha switchable light source is illustrated in the accompanying figures, itis envisioned that the light source can be any of a number of types oflight sources including but not limited to switchable light sources orpassive light sources. Adapter chip 114 can include in-couplingwaveguides 128, out-coupling waveguides 126 and combiners 130 whichcombine the in-coupling and the out-coupling waveguides. Such combiners130 are well known in the art. Substrate 104 can include waveguides 108and sensing sites 112 in relation to waveguides 108. For example sensingsites 112 can be on top of and in optical communication with waveguides108. Detector 106 can include one or more element 116.

In a first embodiment, as shown in FIG. 1A, switchable light source 102is coupled to and is in optical communication with in-couplingwaveguides 128 on adapter chip 114 through optical fibers 118. Opticalfibers 118 can be a set of optical fibers as shown in FIG. 1A.Additionally, detector 106 can be coupled to and in opticalcommunication with out-coupling waveguides 126 on adapter chip 114, forexample, through a second set of optical fibers 118. Adapter chip 114can further be coupled and in optical communication with one or more ofwaveguides 108 at a first edge of substrate 104. Adapter chip 114 ofthis embodiment and all other embodiments can optionally be configuredto provide removable coupling for substrate 104 or other systemcomponents described herein. Likewise substrate 104 can further beconfigured to provide removable coupling with adapter chip 114 or othercomponents of the system.

FIG. 1B illustrates an exemplary scanning sensing system 100 of theinvention including a switchable light source 102, an adapter chip 114,a substrate 104, optical sensing sites 112 and a detector 106. Adapterchip 114 can include in-coupling waveguides 128 and out -couplingwaveguides 126. In this manner Substrate 104 can include in-couplingwaveguides 110, out-coupling waveguides 108, combiners 132 which combinein-coupling 110 and out-coupling waveguides 108 and sensing sites 112,for example on top of and in optical communication with out-couplingwaveguides 108. Detector 106 can include one or more element 116 asdescribed herein.

In a second embodiment, as shown in FIG. 1B, switchable light source 102is coupled to and is in optical communication with in-couplingwaveguides 128 of adapter chip 114 through a set of optical fibers 118.Additionally, detector 106 is coupled to and in optical communicationwith out-coupling waveguides 126 of adapter chip 114 through a secondset of optical fibers 118. Adapter chip 114 is further coupled to and inoptical communication with one or more in-coupling waveguides 110 andwith one or more of out-coupling waveguides 108, for example, at an edgeof substrate 104.

FIG. 1C illustrates an exemplary scanning sensing system 100 of theinvention including a switchable light source 102, two adapter chips114, a substrate 104, optical sensing sites 112 and a detector 106. Theadapter chips include waveguides 134. The substrate includes waveguides108 and sensing sites 112 on top of and in optical communication withthe waveguides.

In a third embodiment, as shown in FIG. 1C, the switchable light source102 is coupled to and is in optical communication with waveguides 134 onone adapter chip 114 through a set of optical fibers 118. Additionally,detector 106 is coupled to and in optical communication with waveguides134 on a second adapter chip 114 through a second set of optical fibers118. The two adapter chips 114 are further coupled and in opticalcommunication with one or more of waveguides 108 at two opposite edgesof the substrate 104. As described herein, detector 106 can include oneor more elements 116.

FIG. 1D illustrates an exemplary scanning sensing system 100 of theinvention including a combined switchable light source and detector chip120, a substrate 104 and optical sensing sites 112. The combinedswitchable light source and detector chip 120 includes light generatorelements 122, detector elements 124, combiners 136, out-couplingwaveguides 138 and in-coupling waveguides 140. Substrate 104 includeswaveguides 108 and optical sensing sites 112 that can be arranged, forexample, on top of and in optical communication with waveguides 108.

In a fourth embodiment, as shown in FIG. 1D, light generator elements122 are coupled to and in optical communication with in-couplingwaveguides 140. Additionally, detector elements 124 are coupled to andin optical communication with out-coupling waveguides 138 on thecombined switchable light source and detector chip 120. Switchable lightsource and detector chip 120 is further coupled to and in opticalcommunication with one or more waveguides 108 at an edge of substrate104.

Although four exemplary embodiments are specifically disclosed herein,it is envisioned that any of a number of other combinations of couplingthe different components/chips disclosed herein at different edges ofthe components/chips are possible. For example, in one embodiment, afirst switchable light source and detector chip is coupled to a firstedge of the substrate and a second switchable light source and detectorchip is coupled at a second edge of the substrate (not shown). It can beunderstood accordingly that the passage of light waves within thedevices and systems described herein, though described in terms of“left” and “right” can be practiced in a variety of directions andorientations based on the flexible arrangements of components providedherein.

As shown in FIGS. 1A to 1D, in some embodiments system 100 can besubstantially planar. For example, switchable light source 102 can be aplanar chip. This chip can be coupled to a planar adapter chip 114 thatis a second chip, that is further coupled to a planar substrate 104 thatis a third chip. In a particular embodiment, as shown in FIG. 1A, system100 is a planar lightwave circuit including three or more coupled planarchips. In one embodiment two chips are integrated into a single chip(e.g., in the switchable light source and detector chip or in an adapterchip and substrate chip). Such a configuration would be useful in a casewhere the substrate chip is reusable and can be effectively used forlong periods of time. One application of such a configuration would bein a system for detecting biological warfare -associated agents. In suchan application it would be advantageous for the system to operate forlong periods of time without a need for replacing the chip. In addition,having two chips integrated on a single substrate solves the problem ofmaintaining the relative alignment of two chips (e.g., an adapter chipand substrate chip).

Where the system is used in biological applications, including but notlimited to detection of biologically active analytes including nucleicacids, proteins or microorganisms, the substrate can be a multi-elementbio-analysis chip.

It is envisioned that an optical sensing site 112 can be associated witheach waveguide 108. It is envisioned that the number of optical sensingsites on a substrate chip can be greater than 10, greater than 100,greater than 200, greater than 1,000, great than 5,000 or greater than10,000. It is further envisioned that the density of optical sensingsites can be greater than 10 per cm², greater than 100 per cm², greaterthan 1,000 per cm² or greater than 10,000 per cm². In one embodiment thedensity of optical sensing sites is greater than 2,000 per cm².

It is envisioned that in any of the embodiments described herein, that afirst light wave generated by the switchable light source in anin-coupling waveguide induces the sensor to transduce an optical signalresulting in a second light wave in an out-coupling waveguide, thesecond light wave being detectable by the detector.

As illustrated in FIG. 1A, in one advantageous embodiment, System 100 isa planar two-dimensional scanning system. System 100 in this embodimentincludes a planar switchable light source 102, for example, a planaroptical switch or an array of switchable lasers, coupled to the plane ofsubstrate 104 through a planar adapter chip 114, to make up, forexample, a bioanalysis chip plane. Furthermore, switchable light source102 can provide a dynamic source of light for selective and programmedexcitation in respect to individual waveguides 108, providing excitationto part or to all of optical sensing sites 112. A dynamic light sourceincludes but is not limited to a tunable wavelength and/or tunablebandwidth light source. Additionally, system 100 of this embodimentprovides for planar collection of the emitted light from all the excitedsensing sites 112 in out-coupling waveguides 108, specifically in theplane of substrate 104, such that the light collection is insubstantially opposite direction to the light produced in in-couplingwaveguides 110.

FIG. 2 is an exemplary illustration of the scanning system of theinvention as part of a working system 201 in a housing 209. While thescanning sensing system illustrated in FIG. 1A-1D is core of the presentinvention, in order to facilitate the operation of this system, one ormore other modules can be included in a working system that includes thescanning sensing system components of the invention.

FIG. 2 illustrates one possible configuration for a working system 201that can include housing 209 for enclosing various modules of workingsystem 201 including but not limited to substrate 204, robotic system203, switchable light source 202, adapter chip 214, multi-elementdetector 206, electronic boards 207 and interface panel 205. Substrate204, switchable light source 202, and multi-element detector 206, arediscussed in detail below.

In regard to housing 209, as shown in FIG. 2, in one embodiment anenclosure or housing 209 holds in place two fixed chips (e.g., of a3-chip architecture), namely, adapter chip 214, switchable light source202 and multi-element detector 206. Accordingly, in this embodimentsubstrate chip 204 is movable in relation to adapter chip 214,switchable light source 202 and multi-element detector 206. Housing 209can include any number of accurately machined parts and or components asdescribed herein, allowing, for example, the relative alignment of the 3optical chips. The working system housing can optionally includetemperature control and vibration isolation for the working system (notshown).

As shown in FIG. 2, working system 201 can further include an X, Y, Z, θrobotic system 203 for positioning substrate 204 as required withinworking system 201. X, Y, Z, θ robotic system 203 can be a translationstage with several degrees of freedom for receiving or acceptingsubstrate 204, holding it in place, and aligning it in relation to therest of working system 201. As desired, at the end of a run X, Y, Z, θrobotic system 203 can eject substrate 204 from working system 201.

It is envisioned that the working system can further include an aligningsystem (not shown). An aligning system can include one or more lightsources, one or more detectors and one or more cameras for activedetection of the position of the substrate of the invention. Based onthe detected position, the aligning system can align the substrate tothe rest of the working system modules, for example, to provide alignedoptical communication between the substrate and the switchable lightsource and detector chip.

As shown in FIG. 2 working system 201 can further include one or moreelectronic boards 207, for example, an electronic driving board and acontrol board. It is envisioned that one or more electronic boards cancontrol all the different parts of the working system. Electronic boards207 can control switchable light source 202 and any other light sourcepresent in the system. Electronic boards 207 can be adapted to read anyor all of the detectors and cameras in working system 201. Electronicboards 207 can further be adapted to drive robotic system 203 andcontrol its motion, and optionally monitor and control temperature indifferent areas of the system. Electronic boards can include logicelements and processors (not shown). It is envisioned that electronicboards can further include embedded software both for controlling theworking system and for interfacing the outside world, for example by wayof interface panel 205 which can include a key-pad or any otherinput/output port.

As shown in FIG. 2 working system 201 can additionally include one ormore interface panel 205. It is anticipated that the system will haveone or more interface panel 205 to allow a user to interface with thesystem and operate it. Interface panels can include any number of inputand output ports well known in the art for connecting the system toother systems or to an external control console (not shown).

FIG. 3A illustrates an exemplary substrate 304 of the system of thefirst embodiment of the current invention further including barriers 311intended to block stray light within the substrate and reduce crosstalkbetween the different elements of the substrate. Barriers 311 can belight absorbing or light reflecting. Barriers 311 can be variouslysized, shaped and positioned between waveguides 308 in any of a numberof orientations to achieve a desired optical effect. As shown in FIG.3A, barriers 311 can be arranged between two adjacent waveguides andproximal to optical sensing sites 312. Waveguides 308 are used to guidea primary light wave (excitation light; see dashed arrow) from the leftedge of substrate 304 to optical sensing sites 312. Waveguides 308 thenguide a secondary light wave (collected at optical sensing sites 312;see dashed arrow) from optical sensing sites 312 back to the left edgeof substrate 304.

FIG. 3B illustrates an exemplary substrate 304 of the system of thesecond embodiment of the current invention further including in-couplingwaveguides 310 and combiners 326. The primary light wave (excitationlight; see dashed arrow) is coupled to substrate 304 through in-couplingwaveguides 310 at the left edge of substrate 304. The excitation lighttraveling from left to right is combined by combiners 326 intoout-coupling waveguides 308 which further guides it to optical sensingsites 312. Out-coupling waveguides 308 are then used to guide thesecondary light wave (collected at sensing sites 312; see dashed arrow)from optical sensing sites 312 back to the left edge of substrate 304.Barriers 311 have the same purpose as described above in FIG. 3A.

FIG. 3C schematically illustrates a cross section of the substrate 304of the current invention. In the example illustrated, in/out couplingwaveguides 308 are embedded underneath a surface of substrate 304.Optical sensing sites 312 can be etched into a surface, for example, theupper cladding of substrate 304 and located, for example, adjacent andon top of waveguide 308 facilitating optical communication between them.It is envisioned that in a different embodiment, the optical sensingsites can also be located on the surface of substrate 304 or etched onlypart of the way into the upper cladding, or all the way through thewaveguides (not shown). It is also envisioned that the waveguides can besingle-mode waveguides, multi-mode waveguides or any combination of thetwo, namely, single mode in the vertical dimension and multi-mode in thelateral dimension.

FIG. 4A illustrates the adapter chip 414 of the first embodiment (seeFIG. 1A) of the current invention. The adapter chip 414 includesin-coupling waveguides 428, out-coupling waveguides 426 and combiner430. A primary light wave (excitation light) can be coupled from leftinto in-coupling waveguides 428. The excitation light is then combinedby combiners 430 into waveguides 426 which then guides it to the rightedge of adapter chip 414 and couples it out to the substrate (notshown). The substrate (not shown) couples back the secondary light wave(collected at the optical sensing sites—not shown) to out-couplingwaveguides 426 at the right edge of adapter chip 414. The secondarylight wave is guided by waveguides 426 from right to left and is coupledout of adapter chip 414 at its left edge to the detector (not shown).

FIG. 4B illustrates adapter chip 414 of the second embodiment (see FIG.1B) of the current invention. Adapter chip 414 includes in-couplingwaveguides 428 and out-coupling waveguides 426. A primary light wave(excitation light) can be coupled from left into in-coupling waveguides428. Waveguide 428 guides the primary light wave to the right edge ofthe adapter chip 414 and couples it out to the substrate (not shown).The substrate (not shown) couples back the secondary light wave(collected at the optical sensing sites—not shown) to out-couplingwaveguides 426 at the right edge of adapter chip 414. The secondarylight wave is guided by waveguides 426 from right to left and is coupledout of adapter chip 414 at its left edge to the detector (not shown).

A range of dimensions for the various features described herein include:waveguides thickness—20 nm to 50 μm; waveguide width—1 μm to 500 μm;waveguide length—1 mm to 100 mm; optical sensing site length—100 μm to100 mm; optical sensing site width—1 μm to 500 μm; optical sensing sitedepth—0 to 20 μm; waveguide pitch—10 μm to 10 mm; substratethickness—100 μm to 5 mm; upper cladding thickness—0 to 20 μm; and lowercladding thickness—0.1 μm to 20 μm.

FIG. 4C in a side view illustrates another embodiment of substrate 404of the invention in relation to a thermal transfer element 403, forexample, a thermoelectric cooler (TEC). Thermal transfer element 403 isa temperature control system useful for heating or cooling a chip, forexample, substrate 404. Although the thermal transfer element may bereferred to herein as a cooling element, it is to be understood thatwhere the thermal transfer element is configured to increase anddecrease the temperature of a chip, the component functions essentiallyas a heating and as a cooling element depending on the induced directionof the electrical current. The thermal transfer element can provide arange of useful temperatures. For example, the thermal transfer elementcan be configured to provide a temperature in the range between about−40° C. to about 120° C. as desired. As illustrated in FIG. 4C, thermaltransfer element 403 can be adapted to receive substrate 404 of theinvention. Thermal transfer element 403 can be adapted to contact partor all of a surface of substrate 404 of the invention.

Providing thermal transfer element 403 in conjunction with substrate 404of the invention is useful, for example, for the amplification of testedsample molecules through processes such as Polymerase Chain Reaction(PCR) as described herein. In use, the embodiment as described for FIG.4C provides the capability of controlling the temperature of the entiresubstrate such that as the temperature of the entire substrate iscycled, samples at any optical sensing site can be amplified by PCRsimultaneously.

FIG. 4D illustrates another embodiment of substrate 404 of the inventionwherein optical sensing site 412 includes heater 405 and thermistor 407.In this embodiment, optical sensing site 412 of substrate 404 caninclude heater 405, for example, a thin-film heater, in the vicinity ofone or more sensing site 412. Heater 405 can be adapted to enableindividual temperature control for each sensing site 412. In addition toheater 405, thermistor 407 can be located at or near one or more sensingsite 412 thereby providing for measuring the local temperature. In use,this embodiment provides the capability of running the same or anydesired different number of cycles and the same or any desired differenttemperature profiles for each and every sensing site.

The adapter of the various embodiments described herein can include aplurality of optical elements comprising at least one lens and at leastone filter, wherein the plurality of optical elements are configured tomanipulate and couple light from the light source to the substrate andfurther configured to manipulate and couple light from the substrate tothe detector. It is envisioned that the optical elements can beindividual elements including lenses and/or filters. It is furtherenvisioned that the optical elements can have a size configuration thatis suitable for interfacing with the light source, substrate, detectorand other elements of the systems described herein.

Advantageously, the embodiments described for FIGS. 4C and 4D cansupport real-time PCR. As described herein, since optical detection isdone from within the substrate, signal detection in both embodiments(see FIGS. 4C and 4D) can be done while the samples are in the processof the amplification cycles, thereby enabling real time analysis of thePCR process.

FIG. 4E illustrates yet another embodiment of substrate 404 of theinvention wherein substrate 404 additionally includes reservoirs 413 andmicrochannels 409 in relation to optical sensing sites 412. As such, inthis embodiment microfluidics are incorporated into the substrate.Microfluidics can be adapted to drive liquid (in this case the testedsample) using the capillary effect across the substrate. As illustratedin FIG. 4E, this can be achieved by an arrangement of microchannels 409,optionally of varying width, which force the sample from one or morereservoirs 413 to optical sensing sites 412 which can include etchedwells to receive the sample. The microchannels can be either etched onthe face of the chip itself or can be added as an external structure ona surface of the substrate 404.

In use, it is envisioned that a sample to be tested can be pipetted intoa reservoir at one end of the substrate. The sample can then bedistributed using the microfluidic system to the optical sensing sitesand sensing wells where it is allowed to bind to pre-spotted probes andcan subsequently be optically scanned and analyzed. Several reservoirsmay be used to separate different samples/patients or for runningseveral parallel tests.

The substrate of the scanning sensing system can made up of any of anumber of well known materials suitable for use in planar lightwavecircuits. For example, useful substrate materials include but are notlimited to Silica (SiO2), glass, epoxy, lithium niobate and indiumphosphide as well as combinations thereof. The waveguides disclosedherein can be made up of Silicon, Silica (SiO2) and derivatives thereof,silicon oxynitride (SiON) and derivatives thereof, silicon nitride (SiN)and derivatives thereof, polymers, lithium niobate and indium phosphideas well as combinations thereof. In one embodiment, UV light is used tochange the refractive index of a waveguide material after deposition.

FIG. 5A illustrates an exemplary silicon layer 520 of the substrate 504.For example, the silicon layer 520 can be made up of a silicon waferhaving a thickness from about 0.1 mm to 10 mm. In another example thesilicon wafer can have a thickness from about 0.3 to 1 mm. In aparticular example as illustrated in FIG. 5A, the silicon wafer has athickness of 0.65 mm. As shown in FIG. 5A in one embodiment, the silica(SiO2) layer 522 is a 14 μm thermal oxide layer of Silica (SiO2) createdby placing the Silicon in an oxygen-rich environment inside a furnace athigh temperature. The top Silicon layer oxidizes over time (severalhours) creating a SiO2 layer. Additionally, as shown in FIG. 5A, in oneembodiment, the cladding layer 524 is 15 μm thick and deposited by aPECVD (Plasma-Enhanced Chemical Vapor Deposition) process after etchingto produce the waveguides 508.

It is envisioned that the various layers of the substrate can includedifferent refraction index properties. For example, a waveguide layer(e.g. SiN) has a higher refraction index than a cladding layer of silicadeposited thereon.

As shown in FIG. 5B (illustrated with a photomicrograph prior todeposition of a cladding layer), in some embodiments, the substrate 504can include two waveguides 508 arranged for light wave coupling on asilica (SiO2) layer 522. Alternatively, as shown in FIG. 5C, twowaveguides 508 can be arranged for guiding uncoupled light waves on asilica (SiO2) layer 522 and over-clad with a cladding layer 524.

The optical sensing sites in one embodiment are in the form of wells,for example, etched wells (see FIG. 3C cross-section view). Where theoptical sensing site is a well, it can act as a vessel for a liquidsample. In another embodiment the optical sensing sites are a region onthe surface of the substrate, for example, above the waveguides. In afurther embodiment, the optical sensing sites are biochemicalinteraction sites. For example, where the optical sensing site is a wellcontaining a sensor single stranded DNA oligonucleotide having afluorescent tag attached, a solution containing a target complementarysingle stranded DNA added to the well could biochemically interact bybase-pairing with the sensor within the optical sensing site (notshown). In another example the optical sensing site is a location orwell containing one or more immunoassay reagent for conducting animmunoassay as described herein.

In a particular embodiment, the optical sensing sites comprise opticaltransducers (not shown). An optical transducer is defined as any devicethat generates a measurable change (wavelength, amplitude or phase) tothe incoming primary light wave and can thus be monitored in theoutgoing secondary light wave. In one embodiment the optical transducersare fluorescence wells including fluorescent or luminescent compounds,wherein light waves guided by the waveguides excite the fluorescent orluminescent compound in the wells in the presence of a target, and thesame waveguides collect and guide light emitted from the wells to thedetector (through the adapter chip), for example at the edge of the chip(not shown).

The sensor of the optical sensing site of the system can be a sensorthat discriminates or interacts with a target (e.g., a biologicallyactive analyte) in a sample from, for example, a biological, man-made orenvironmental source. As discussed above, a first lightwave can inducethe sensor to transduce an optical signal to a second light wave. In oneembodiment where the sensor is capable of discriminating or interactingwith a target in a sample, a measurable change in the second light wavecan result when the sensor discriminates or interacts with the target.Upon detection of the change in the second light wave using the detectorof the system, presence of the target in the sample is known.

Any of a number of sensors can be used with the scanning sensing systemto measure phenomenon associated with sensing of a target in a sample.Examples of suitable sensors include, but are not limited to, afluorescence well or cell, an absorption cell, an interferometricsensor, a diffractive sensor or a Surface Plasmon Resonance (SPR)detector. For a fluorescence well or cell, the phenomenon measurable canbe light emission from luminescent or fluorescent molecular tags. Forexample, emitted light at an altered wavelength can be measured. In thecase of an absorption cell, changes in the sample optical density (OD)can measurably affect the intensity of the light passing through thesample. For an interferometric sensor, changes in the effectiverefractive index of a waveguide generate a phase different between twolight waves leading to different interference patterns measurable as adifference in intensity at the detector. For a diffractive sensor,changes in the effective refractive index at the surface of adiffractive element, for example, a grating, affect the diffractionangle of the light for a given wavelength or alternatively affect thewavelength at a given diffraction angle. In the case of a SPR sensor,changes in the effective refractive index at a metal-dielectricinterface affect the resonance conditions for generating surfacePlasmons.

FIG. 6A illustrates an exemplary switchable light source 602 of thesystem of the invention, including one or more inputs 601 as a primarysource of light for coupling to a light generator. The light generatorcan be any source of electromagnetic radiation emitting one or morediscrete spectral-lines or a continuous spectrum (not shown). In onepreferred embodiment the light generator is a laser source emitting inone or more well defined wavelengths. In a second preferred embodimentthe light generator is a tunable laser that can be tuned to emit lightin one wavelength within a predefined range. As illustrated, switchablelight source 602 further includes a plurality of outputs 603 shown inFIG. 6A as N—Outputs. The number of outputs 603 included in switchablelight source 602 can be variable based on the intended use. For example,in certain applications the number of outputs 603 can be greater than 10outputs. In one embodiment the number of outputs 603 can be great than100 outputs. In a further embodiment the number of outputs 603 can begreater than 1,000 outputs. In another embodiment the number of outputs603 ranges from about 50 to 500.

The light source can be a passive lxN splitter with N being for example,between 1 and 1,000. It is further envisioned that N can be greater than1,000, greater than 10,000 or greater than 100,000. Such an arrangementis advantageous in that is allows for simultaneous (e.g. parallel)excitation in waveguides of the system as described herein.

In a particular embodiment, the number of outputs 603 is about 128. Asshown in FIG. 6A, in one embodiment, the switchable light sourceincludes outputs 603 that fan out from an input 601 equally splittingthe light at input 601 to all outputs 603. As illustrated in FIG. 6B, inone embodiment a branched architecture stemming from the input 601 tothe outputs 603 can be used. Although only one input is shown in FIGS.6A and 6B, it is envisioned that multiple inputs 601 can be used.

FIG. 6C illustrates an exemplary switchable light source and detectorchip 620 of one system of the invention including light generatorelements 622, detector elements 624, in -coupling waveguides 640,out-coupling waveguides 638 and combiners 636. Light generator elements622 generate a primary light wave which is coupled to in-couplingwaveguides 640. The light wave propagates from left to right and iscombined by combiners 636 out-coupling waveguides 638. At the right edgeof switchable light source and detector chip 620 the primary light wavecouples out to the substrate (not shown). A secondary light wavegenerated at the optical sensing sites on the substrate (not shown) iscoupled back to switchable light source and detector chip 620 at itsright edge and propagates from right to left guided by out-couplingwaveguides 638 and into detector elements 624. In this embodiment lightgenerator elements 622 and detector elements 624 are connected through,for example, electronic leads (not shown) to an external electroniccontrol and driver board (not shown) which controls and drives lightgenerator elements 622 and detector elements 624.

It is envisioned that the switchable light source can be a dynamic lightsource allowing for selective and programmed generation of the primarylight wave through one or more individual output. In one embodiment theswitchable light source is an optical switch, for example, a planaroptical switch. The switchable light source can be a light manipulatingdevice for switching light from a given input to any given output.Moreover, the switchable light source can multicast an input light toseveral outputs all at the same time. In one embodiment, switchablelight source is an optical switch coupled to a light generator throughone or more optical fiber (not shown). In a particular embodiment, thelight generator is coupled to one or more of the inputs of theswitchable light source. By way of non-limiting examples, the lightgenerator can provide variable wavelengths of light. In one embodiment,the light generator is a broad-band source. In another embodiment, thelight generator is a tunable source.

The switchable light source can include K (=1, 2, 3 . . . ) inputs and Noutput. In some embodiments, the number of outputs will be equal to thenumber of in-coupling waveguides in the substrate of the system. In aparticular embodiment, the interface between a light generating sourceand the switchable light source inputs includes optical fibers. Theinterface between the switchable light source and detector chip outputsshould match, in terms of pitch, the in-coupling waveguides in thesubstrate to allow these two elements to butt-couple and transfer lightfrom the switchable light source and detector chip to the in-couplingwaveguides on the substrate.

In one embodiment the optical switch includes individual switchingelements based on Mach Zehnder interferometers.

The light source and detector chip can include an array of lightgenerator elements. In one implementation, the light generator elementsare light emitting diodes (LED). In another implementation the lightgenerator elements are laser chips. Each individual light generatorelement is separately controlled and can be turned on or off as desired.In one implementation the light source and detector chip includes 10 ormore light generator elements. In another implementation the lightsource and detector chip includes 100 or more light generator elements.In yet another implementation the light source and detector chipincludes 1000 or more light generator elements. In a particularimplementation the light source and detector chip includes between 10and 100 light generator elements.

The light source and detector chip can include an array of detectorelements. In one implementation, the detector elements are PIN diodes.In another implementation the detector elements are AvalanchePhoto-Diodes (APD). Each individual detector element is separatelycontrolled and read. In one implementation the light source and detectorchip includes 10 or more detector elements. In another implementationthe light source and detector chip includes 100 or more detectorelements. In yet another implementation the light source and detectorchip includes 1000 or more detector elements. In a particularimplementation the light source and detector chip includes between 10and 100 detector elements.

The light generator elements array on the light source and detector chipcan be integrated on a single chip with the detector elements array.Such a chip includes an array of two or more light generator elements,an array of two or more detector elements, an array of two or morein-coupling waveguides, an array of two or more out-coupling waveguidesand an array of two or more combiners. In one implementation each lightgenerator element is optically coupled to one in-coupling waveguide andadapted such that most of the light emitted by the light generatorelement propagates along that waveguide. The waveguides can extend tothe edge of the chip where they can be brought to couple the lightpropagating within them to the substrate. In one implementation twolight generator elements, each optionally emitting at a differentwavelength can be coupled to a single in-coupling waveguide. In anotherimplementation more than two light generator elements, each optionallyemitting at a different wavelength can be coupled to a singlein-coupling waveguide.

The light source and detector chip can include in addition to a lightgenerator elements, detector elements and waveguides, light manipulatingfeatures such as filters, switches, modulators, splitters, combiners,mirrors and circulators.

The control of the light source and detector chip can be eitherintegrated on the same chip as the light generator elements, detectorelements and waveguides or alternatively can be external to the chip.The light source and detector chip can have an electrical interface toan external driver or external controller or logic interface to anexternal control system. The control of the light source and detectorchip allows driving each light generator element and each detectorelement separately. It further allows also control of the other featurespresent on the light source and detector chip such as, for example, themodulators and switches.

The light source and detector chip can couple to the substrate inseveral different ways. In one implementation the coupling is done bybringing the ends of the waveguides on both chips (the light source anddetector chip and the sensing substrate) in close proximity and allowingthe light to flow directly from one waveguide to the other. In anotherimplementation, a portion of the waveguides on both chips are aligned ontop of each other, parallel and in close proximity to each other, thuscoupling light from one waveguide to the other through the evanescentelectromagnetic field.

All coupling schemes described between the light source and detectorchip and the sensing substrate apply to the coupling between the adapterchip and the sensing substrate.

Additional elements useful in planar lightwave circuits, including butare not limited to couplers, filters, mirrors, circulators, splitters,modulators, switches and trenches are envisioned as part of the systemdescribed herein (not shown). Such elements when integrated into thesensing substrate or into the light source and detector chip can serveto manipulate the incoming first light waves in the in-couplingwaveguides or outgoing second light waves in the out-couplingwaveguides.

FIG. 7 illustrates an exemplary detector 706 of the system of theinvention including elements 716 (shown as M-elements). In oneembodiment, as shown in FIG. 7, the detector 706 includes an array oflight sensitive elements 716, for example, in the form of aphotodetector array. In one embodiment, as shown in FIG. 1A, the numberof elements 116 matches the number of out-coupling waveguides 126 in theadapter chip 114.

In one non-limiting example, the detector is a detector array having aspectral range of between 400 to 1000 nm, a photosensitivity (A/W)of >0.3, an active area per element of 0.005 mm², 128 elements, and apitch of <0.1 mm.

In one embodiment, the detector is a silicon photodiode (PN, PIN or APD)array. An example of a suitable detector array is the Texas AdvancedOptoelectronic Solutions (TAOS) 1×128 linear array (PN-TSL1210R).

A control system for managing the different steps of operating thescanning sensing system is envisioned.

The control system can manage steps such as alignment of the lightsource and detector chip, sensing substrate and adapter chip, inaddition to switching the light output from the light source, readingthe detector array and reporting the results detected.

In practicing the methods of the present invention, many conventionaltechniques in molecular biology are optionally utilized. Thesetechniques are well known and are explained in, for example, Ausubel etal. (Eds.) Current Protocols in Molecular Biology, Volumes I, II, andIII, (1997), Ausubel et al. (Eds.), Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, 5th Ed., John Wiley & Sons, Inc. (2002), Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring HarborLaboratory Press (2000), and Innis et al. (Eds.) PCR Protocols: A Guideto Methods and Applications, Elsevier Science & Technology Books (1990),all of which are incorporated herein by reference.

Sample preparation suitable for use with the system and methodsdescribed herein can include any of a number of well know methods forcollection and analysis of biological and/or environmental samples. Inthe case of biological samples the sample can be, for example,manipulated, treated, or extracted to any desired level of purity for atarget of interest.

The sample can be bodily fluids suspected to contain a biologicallyactive analyte. Commonly employed bodily fluids include but are notlimited to blood, serum, saliva, urine, gastric and digestive fluid,tears, stool, semen, vaginal fluid, interstitial fluids derived fromtumorous tissue, and cerebrospinal fluid.

It is anticipated that the systems described herein can be used forscreening a large variety of samples. In the case where the investigatedsubject is a living creature, the sample may originate from body fluidsas discussed. Methods of obtaining samples include but are not limitedto cheek swabbing, nose swabbing, rectal swabbing, skin fat extractionor other collection strategies for obtaining a biological or chemicalsubstance. When the tested subject is a non-living or environmentalbody, the sample may originate from any substance in a solid phase,liquid phase or gaseous phase. The sample may be collected and placedonto the sensing substrate or the sensing substrate may be directlyexposed to the investigated sample source (e.g. water reservoir, freeair) and interact with it.

In some embodiments, the bodily fluids are used directly for detectingone or more biologically active analyte present therein with the subjectscanning sensing device without further processing. Where desiredhowever, the bodily fluids can be pre-treated before performing theanalysis with the subject scanning sensing devices. The choice ofpre-treatments will depend on the type of bodily fluid used and/or thenature of the biologically active analyte under investigation. Forinstance, where the biologically active analyte is present at low levelin a sample of bodily fluid, the sample can be concentrated via anyconventional means to enrich the biologically active analyte. Methods ofconcentrating a biologically active analyte include but are not limitedto drying, evaporation, centrifugation, sedimentation, precipitation,and amplification. Where the biologically active analyte is a nucleicacid, it can be extracted using various lytic enzymes or chemicalsolutions according to the procedures set forth in Sambrook et al.

(“Molecular Cloning: A Laboratory Manual”), or using nucleic acidbinding resins following the accompanying instructions provided bymanufactures. Where the biologically active analyte is a moleculepresent on or within a cell, extraction can be performed using lysingagents including but not limited to denaturing detergent such as SDS ornondenaturing detergent such as thesit (2-dodecoxyethanol), sodiumdeoxylate, Triton® X-100, and Tween® 20.

In some embodiments, pretreatment can include diluting and/or mixing thesample, and filtering the sample to remove, e.g., red blood cells from ablood sample.

Targets detectable using the scanning sensing system include but are notlimited to, a biologically active analyte including a nucleic acid, aprotein, an antigen, an antibody, a microorganism, a gas, a chemicalagent and a pollutant.

In one embodiment, the target is a nucleic acid that is DNA, forexample, cDNA. In a related embodiment, the DNA target is produced viaan amplification reaction, for example, by polymerase chain reaction(PCR). In another embodiment of the subject invention, the detectedbiologically active analyte is a protein representing a known biomarkerfor a disease or specific condition of the investigated organism. Inanother embodiment several different biologically active analytes can beproteins provided as a panel of bio-markers wherein relativeconcentrations of the bio-markers are indicative for a disease or othercondition of the investigated organism. In a further embodiment thetarget is a microorganism that is a pathogen. In another embodiment thetarget is a chemical agent, for example, a toxic chemical agent.

Where the target is a nucleic acid, it can be single-stranded,double-stranded, or higher order, and can be linear or circular.Exemplary single-stranded target nucleic acids include mRNA, rRNA, tRNA,hnRNA, ssRNA or ssDNA viral genomes, although these nucleic acids maycontain internally complementary sequences and significant secondarystructure. Exemplary double-stranded target nucleic acids includegenomic DNA, mitochondrial DNA, chloroplast DNA, dsRNA or dsDNA viralgenomes, plasmids, phage, and viroids. The target nucleic acid can beprepared synthetically or purified from a biological source. The targetnucleic acid may be purified to remove or diminish one or more undesiredcomponents of the sample or to concentrate the target nucleic acids.Conversely, where the target nucleic acid is too concentrated for theparticular assay, the target nucleic acid may be diluted.

Following sample collection and optional nucleic acid extraction, thenucleic acid portion of the sample comprising the target nucleic acidcan be subjected to one or more preparative reactions. These preparativereactions can include in vitro transcription (IVT), labeling,fragmentation, amplification and other reactions. mRNA can first betreated with reverse transcriptase and a primer to create cDNA prior todetection and/or amplification; this can be done in vitro with purifiedmRNA or in situ, e.g. in cells or tissues affixed to a slide. Nucleicacid amplification increases the copy number of sequences of interestsuch as the target nucleic acid. A variety of amplification methods aresuitable for use, including the polymerase chain reaction method (PCR),the ligase chain reaction (LCR), self sustained sequence replication(3SR), nucleic acid sequence-based amplification (NASBA), the use of QBeta replicase, reverse transcription, nick translation, and the like.

Where the target nucleic acid is single-stranded, the first cycle ofamplification forms a primer extension product complementary to thetarget nucleic acid. If the target nucleic acid is single stranded RNA,a polymerase with reverse transcriptase activity is used in the firstamplification to reverse transcribe the RNA to DNA, and additionalamplification cycles can be performed to copy the primer extensionproducts. The primers for a PCR must, of course, be designed tohybridize to regions in their corresponding template that will producean amplifiable segment; thus, each primer must hybridize so that its 3′nucleotide is paired to a nucleotide in its complementary templatestrand that is located 3′ from the 3′ nucleotide of the primer used toreplicate that complementary template strand in the PCR.

The target nucleic acid can be amplified by contacting one or morestrands of the target nucleic acid with a primer and a polymerase havingsuitable activity to extend the primer and copy the target nucleic acidto produce a full length complementary nucleic acid or a smaller portionthereof. Any enzyme having a polymerase activity that can copy thetarget nucleic acid can be used, including DNA polymerases, RNApolymerases, reverse transcriptases, enzymes having more than one typeof polymerase activity, and the enzyme can be thermolabile orthermostable. Mixtures of enzymes can also be used. Exemplary enzymesinclude: DNA polymerases such as DNA Polymerase I (“Pol I”), the Klenowfragment of Pol I, T4, T7, Sequenase® T7, Sequenase® Version 2.0 T7,Tub, Tag, Tth, Pfx, Pfu, Tsp, Tfl, Tli and Pyrococcus sp GB D DNApolymerases; RNA polymerases such as E. coli, SP6, T3 and T7 RNApolymerases; and reverse transcriptases such as AMV, M MuLV, MMLV, RNAseH′ MMLV (Superscript®), Superscript® II, ThermoScript®, HIV 1, and RAV2reverse transcriptases. All of these enzymes are commercially available.Exemplary polymerases with multiple specificities include RAV2 and Tli(exo) polymerases. Exemplary thermostable polymerases include Tub, Taq,Tth, Pfx, Pfu, Tsp, Tfl, Tli and Pyrococcus sp. GB D DNA polymerases.

Suitable reaction conditions are chosen to permit amplification of thetarget nucleic acid, including pH, buffer, ionic strength, presence andconcentration of one or more salts, presence and concentration ofreactants and cofactors such as nucleotides and magnesium and/or othermetal ions (e.g., manganese), optional cosolvents, temperature, thermalcycling profile for amplification schemes comprising a polymerase chainreaction, and may depend in part on the polymerase being used as well asthe nature of the sample. Cosolvents include formamide (typically atfrom about 2 to about 10%), glycerol (typically at from about 5 to about10%), and DMSO (typically at from about 0.9 to about 10%). Techniquesmay be used in the amplification scheme in order to minimize theproduction of false positives or artifacts produced duringamplification. These include “touchdown” PCR, hot start techniques, useof nested primers, or designing PCR primers so that they form stem-loopstructures in the event of primer-dimer formation and thus are notamplified. Techniques to accelerate PCR can be used, for example,centrifugal PCR, which allows for greater convection within the sample,and comprising infrared heating steps for rapid heating and cooling ofthe sample. One or more cycles of amplification can be performed. Anexcess of one primer can be used to produce an excess of one primerextension product during PCR; preferably, the primer extension productproduced in excess is the amplification product to be detected. Aplurality of different primers may be used to amplify different targetnucleic acids or different regions of a particular target nucleic acidwithin the sample.

Amplified target nucleic acids may be subjected to post amplificationtreatments. For example, in some cases, it may be desirable to fragmentthe target nucleic acid prior to hybridization in order to providesegments which are more readily accessible. Fragmentation of the nucleicacids can be carried out by any method producing fragments of a sizeuseful in the assay being performed; suitable physical, chemical andenzymatic methods are known in the art.

An amplification reaction can be performed under conditions which allowa nucleic acid associated with the optical sensing site to hybridize tothe amplification product during at least part of an amplificationcycle. When the assay is performed in this manner, real time detectionof this hybridization event can take place by monitoring for lightemission during amplification.

Real time PCR product analysis (and related real timereverse-transcription PCR) provides a well-known technique for real timePCR monitoring that has been used in a variety of contexts, which can beadapted for use with the methods described herein (see, Laurendeau etal. (1999) “TaqMan PCR-based gene dosage assay for predictive testing inindividuals from a cancer family with INK4 locus haploinsufficiency”Clin Chem 45(7):982-6; Bièche et al. (1999) “Quantitation of MYC geneexpression in sporadic breast tumors with a real-time reversetranscription-PCR assay” Cancer Res 59(12):2759-65; and Kreuzer et al.(1999) “LightCycler technology for the quantitation of bcr/abl fusiontranscripts” Cancer Res 59(13):3171-4, all of which are incorporated byreference). In addition, linear PCR and Linear-After-The Exponential(LATE)-PCR can be adapted for use with the methods described herein.

Immunoassays can be conducted on the scanning sensor system of theinvention, for example, at one or more optical sensing site of thesystem. Suitable immunoassay systems include but are not limited tocompetitive and noncompetitive assay systems. Such assay systems aretypically used with techniques such as western blots, radioimmunoassays,EIA (enzyme immunoassay), ELISA (enzyme-linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, protein. A immunoassays, and cellularimmunostaining (fixed or native) assays to name but a few. Such assaysare routine and well known in the art (see, e.g., Ausubel et al.,supra). Immunoassay techniques particularly useful with the scanningsensor systems described herein include but are not limited to ELISA,“sandwich” immunoassays, and fluorescent immunoassays. Exemplaryimmunoassays are described briefly below (but are not intended by way oflimitation).

ELISAs generally involve preparing antigen, coating a well (e.g., anoptical sensing site of the scanning sensor system) with the antigen,adding the antibody of interest conjugated to a detectable compound suchas an enzymatic substrate (e.g., horseradish peroxidase or alkalinephosphatase) to the well and incubating for a period of time, anddetecting the presence of the antigen. In ELISAs the antibody ofinterest does not have to be conjugated to a detectable compound;instead, a second antibody (which recognizes the antibody of interest)conjugated to a detectable compound may be added to the well. Further,instead of coating the well with the antigen, the antibody may be coatedto the well. In this case, a second antibody conjugated to a detectablecompound may be added following the addition of the antigen of interestto the coated well. One of skill in the art would be knowledgeable as tothe parameters that can be modified to increase the signal detected aswell as other variations of ELISAs known in the art.

In one exemplary immunoassay, a sample contains an unknown amount ofbiologically active analyte to be measured, which may be, for example, aprotein. The analyte may also be termed an antigen. The sample may bespiked with a known or fixed amount of labeled analyte. The spikedsample is then incubated with an antibody that binds to the analyte, sothat the analyte in the sample and the labeled analyte added to thesample compete for binding to the available antibody binding sites. Moreor less of the labeled analyte will be able to bind to the antibodybinding sites, depending on the relative concentration of the unlabeledanalyte present in the sample. Accordingly, when the amount of labeledanalyte bound to the antibody is measured, it is inversely proportionalto the amount of unlabeled analyte in the sample. The amount of analytein the original sample may then be calculated based on the amount oflabeled analyte measured, using standard techniques in the art.

In one exemplary competitive immunoassay, an antibody that binds to abiologically active analyte may be coupled with or conjugated with aligand, wherein the ligand binds to an additional antibody added to thesample being tested. One example of such a ligand includes fluorescein.The additional antibody may be bound to a solid support (e.g., anoptical sensing site of the scanning sensor system). The additionalantibody binds to the ligand coupled with the antibody that binds inturn to the analyte or alternatively to the labeled analyte, forming amass complex which allows isolation and measurement of the signalgenerated by the label coupled with the labeled analyte.

In another type of exemplary competitive immunoassay, the biologicallyactive analyte to be measured may be bound to a solid support (e.g., anoptical sensing site of the scanning sensor system), and incubated withboth an antibody that binds to the analyte and a sample containing theanalyte to be measured. The antibody binds to either the analyte boundto the solid support or to the analyte in the sample, in relativeproportions depending on the concentration of the analyte in the sample.The antibody that binds to the analyte bound to the solid support isthen bound to another antibody, such as anti-mouse IgG, that is coupledwith a label. The amount of signal generated from the label is thendetected to measure the amount of antibody that bound to the analytebound to the solid support. Such a measurement will be inverselyproportional to the amount of analyte present in the sample. Such anassay may be used in the scanning sensor system of the presentinvention.

A wide diversity of labels are available in the art that can be employedfor conducting the subject assays. In some embodiments labels aredetectable by spectroscopic, photochemical, biochemical, immunochemical,or chemical means. For example, useful nucleic acid labels includefluorescent dyes, enzymes, biotin, dioxigenin, or haptens and proteinsfor which antisera or monoclonal antibodies are available. A widevariety of labels suitable for labeling biological components are knownand are reported extensively in both the scientific and patentliterature, and are generally applicable to the present invention forthe labeling of biological components. Suitable labels include enzymes,substrates, cofactors, inhibitors, fluorescent moieties,chemiluminescent moieties, or bioluminescent labels. Labeling agentsoptionally include, for example, monoclonal antibodies, polyclonalantibodies, proteins, or other polymers such as affinity matrices,carbohydrates or lipids. Detection proceeds by any of the methodsdescribed herein, for example, by detecting an optical signal in anoptical waveguide. A detectable moiety can be of any material having adetectable physical or chemical property. Such detectable labels havebeen well-developed in the field of gel electrophoresis, columnchromatography, solid substrates, spectroscopic techniques, and thelike, and in general, labels useful in such methods can be applied tothe present invention. Preferred labels include labels that produce anoptical signal. Thus, a label includes without limitation anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical, thermal, or chemical means.

In some embodiments the label is coupled directly or indirectly to amolecule to be detected such as a product, substrate, or enzyme,according to methods well known in the art. As indicated above, a widevariety of labels are used, with the choice of label depending on thesensitivity required, ease of conjugation of the compound, stabilityrequirements, available instrumentation, and disposal provisions. Nonradioactive labels are often attached by indirect means. Generally, aligand molecule is covalently bound to a polymer. The ligand then bindsto an anti ligand molecule which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. A number ofligands and anti-ligands can be used. Where a ligand has a naturalanti-ligand, for example, biotin, thyroxine, and cortisol, it can beused in conjunction with labeled, anti-ligands. Alternatively, anyhaptenic or antigenic compound can be used in combination with anantibody.

In some embodiments the label can also be conjugated directly to signalgenerating compounds, for example, by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,and umbelliferone. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, such as luminol.

Methods of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence by, for example, ascanning sensor system as described herein. Similarly, enzymatic labelsare detected by providing appropriate substrates for the enzyme anddetecting the resulting reaction product (e.g., a reaction productcapable of producing a detectable optical signal).

In some embodiments the detectable signal may be provided byluminescence sources. “Luminescence” is the term commonly used to referto the emission of light from a substance for any reason other than arise in its temperature. In general, atoms or molecules emit photons ofelectromagnetic energy (e.g., light) when they move from an “excitedstate” to a lower energy state (usually the ground state); this processis often referred to as “radioactive decay”. There are many causes ofexcitation. If the exciting cause is a photon, the luminescence processis referred to as “photoluminescence”. If the exciting cause is anelectron, the luminescence process is referred to as“electroluminescence”. More specifically, electroluminescence resultsfrom the direct injection and removal of electrons to form anelectron-hole pair, and subsequent recombination of the electron-holepair to emit a photon. Luminescence which results from a chemicalreaction is usually referred to as “chemiluminescence”. Luminescenceproduced by a living organism is usually referred to as“bioluminescence”. If photoluminescence is the result of a spin allowedtransition (e.g., a single-singlet transition, triplet-triplettransition), the photoluminescence process is usually referred to as“fluorescence”. Typically, fluorescence emissions do not persist afterthe exciting cause is removed as a result of short-lived excited stateswhich may rapidly relax through such spin allowed transitions. Ifphotoluminescence is the result of a spin forbidden transition (e.g., atriplet-singlet transition), the photoluminescence process is usuallyreferred to as “phosphorescence”. Typically, phosphorescence emissionspersist long after the exciting cause is removed as a result oflong-lived excited states which may relax only through suchspin-forbidden transitions. A “luminescent label” may have any one ofthe above-described properties.

Suitable chemiluminescent sources include a compound which becomeselectronically excited by a chemical reaction and may then emit lightwhich serves as the detectible signal or donates energy to a fluorescentacceptor. A diverse number of families of compounds have been found toprovide chemiluminescence under a variety of conditions. One family ofcompounds is 2,3-dihydro-1,4-phthalazinedione. A frequently usedcompound is luminol, which is a 5-amino compound. Other members of thefamily include the 5-amino-6,7,8-trimethoxy- and thedimethylamino[ca]benz analog. These compounds can be made to luminescewith alkaline hydrogen peroxide or calcium hypochlorite and base.Another family of compounds is the 2,4,5-triphenylimidazoles, withlophine as the common name for the parent product. Chemiluminescentanalogs include para-dimethylamino and -methoxy substituents.Chemiluminescence may also be obtained with oxalates, usually oxalylactive esters, for example, p-nitrophenyl and a peroxide such ashydrogen peroxide, under basic conditions. Other useful chemiluminescentcompounds that are also known include -N-alkyl acridinum esters anddioxetanes. Alternatively, luciferins may be used in conjunction withluciferase or lucigenins to provide bioluminescence.

In a separate embodiment, the present invention provides a method ofmonitoring one or more pharmacological parameter, for example,Pharmacodynamic (PD) and/or pharmacokinetic (PK) parameters, useful forassessing efficacy and/or toxicity of a therapeutic agent. The methodcomprises subjecting a sample of bodily fluid from a subjectadministered with the therapeutic agent to a scanning sensing device formonitoring the one or more pharmacological parameter, the scanningsensing device can be used as described herein to yield detectablesignals indicative of the values of the more than one pharmacologicalparameter from the sample; and detecting the detectable signal generatedfrom said sample of bodily fluid.

In one implementation the samples tested can include a large number of avariety of small molecules (e.g., screening libraries) which are ofinterest when investigating new drugs. Accordingly, the scanning sensingsystem described herein is useful for screening libraries of smallmolecules to investigate their ability to interact with certainbiologically active analytes may reveal potential new drugs. Furtherscreening of some or all small molecule candidates may reveal adversedrug effects and toxicity.

In one implementation the samples can include molecules which are testedfor toxicity.

In general in another aspect methods of using the scanning sensingsystems described herein are provided.

In one embodiment, the light source, for example, an optical switch oran array of light generator elements, couples light into one or morein-coupling waveguides at any given time. The light travels along thewaveguides, reaches the optical sensing sites and interacts through thesensor, for example, an optical transducer. The samples are positionedat or near the waveguides. Next, the light leaving the sensor couplesinto the out-coupling waveguides and travels down the waveguide to itsend at an edge of the substrate, for example, a chip facet. Lightexiting the out-coupling waveguides is then detected by the differentelements of the detector, which can be a detector array.

In another embodiment, scanning sensing of a sample includes deliveringa sample suspected of containing a target to be detected to an opticalsensing site of the scanning sensor system. Delivering a sample to thesystem can include pipetting of a fluid to the optical sensing site.Other delivery means can include but are not limited to robotic fluiddelivery system or physically depositing a non-fluid or semi-fluidsample at the optical sensing site, either by hand or with the aid of atool or robot manipulation system. Next, a first light wave produced bythe light source is provided to one or more of the plurality ofwaveguides in optical communication with the optical sensing site. Thefirst light wave is transduced (e.g., measurably changed) by the sensorassociated with the optical sensing site to form a second light wavecarried back in one or more of the plurality of out-coupling waveguideswhich are in optical communication with the optical sensing site. Next ameasurable change in the second light wave is detected using thedetector which is in optical communication with the out-couplingwaveguides. Detection of measurable change in the second light wavesindicates that the sensor has interacted with the target. It isenvisioned that the waveguides described herein can be arrangedsubstantially parallel as illustrated generally in the accompanyingfigures.

In a further embodiment, scanning sensing includes switching one or morelight wave from the light source into the substrate to produce the firstlight wave in one or more of the waveguides in a controlled and scanningmanner.

In another embodiment, the light source includes an optical switch forcontrolling switching of one or more input light wave. The opticalswitch can multicast light to a plurality of outputs. The plurality oflight waves can be coupled into the sensing substrate to controllablyproduce the first light wave in one or more of the waveguides.

In one embodiment, all in-coupling waveguides are provided with a firstlight wave and simultaneous detection of second light waves at eachout-coupling waveguide is achieved using a detector that is aphotodetector array.

By switching light between waveguides, each waveguide can beindividually addressed with a first light wave. The order of addressingthe waveguides can be sequential, staggered, random or in any orderdesired. Rapid scanning of the entire array of optical sensing sites canbe achieved with the aid of the photodetector array since any secondlight wave associated with each out-coupling waveguide can besimultaneously detected.

In various embodiments the method of using the scanning sensing systeminvolves the detection of a substance, including but not limited to abiologically active analyte including a nucleic acid, a protein, anantigen, an antibody, a panel of proteins, a microorganism, a gas, achemical agent and a pollutant. In a particular embodiment, a singlenucleotide polymorphism (SNP) is detected in the target. In oneembodiment expression of a gene is detected upon detection of thetarget.

Systems using planar waveguides for optical detection of SNPs have beendescribed before. For example, single base extension (“SBEX”) withplanar waveguide fluorescent biosensor technology to detect SNPs hasbeen described by Herron and Tolley in U.S. patent application Ser. No.10/984,629, filed Nov. 8, 2004 and titled “Single Base Extension.”Briefly, total internal reflectance fluorometry (TIRF) can be used incombination with SBEX under real time detection conditions for SNPdetection using planar waveguide technology. Evanescent waves generatedin a waveguide substrate will only excite fluorescently labeled analyteDNA molecules that are bound to stationary capture oligonucleotides.Herron found that the depth of evanescent wave useful for measurementsis within about 300 nm of the sensor surface. The SBEX approach uses aDNA polymerase to incorporate, for example, Cy5 labeleddideoxynucleotriphosphates (ddNTPs). Additional labels are discussedelsewhere herein.

Identification (“calling”) of the single base added to the 3′ end of theprobe molecule can be done in one of three ways: parallel channels foreach of the four bases using a different labeled ddNTP in each channel;sequential SBEX reactions using a different labeled ddNTP in eachreaction; or wavelength discrimination of the four possibilities using adifferent fluorescent label for each ddNTP. The first of these methodsmay be preferred. SBEX may be used in oligonucleotide genotyping and SNPdetection systems, and is advantageous over traditional hybridizationassays, for example, due to greater base specificity, production of acovalent bond between the labeled ddNTP and the probe, and simultaneousdetection of multiple bases.

By using SBEX on waveguides, simultaneous detection of several differentpolymorphisms can be done with ease. By patterning the waveguide withdifferent capture sequences, different points in a sequence, forexample, a genome, a chromosome and/or a gene, may be assayed. As SBEXonly requires a fluorescent label on the ddNTP monomers used, allinstances of a particular base will be detected. In order to do the samething with a traditional DNA hybridization assay, each probe DNA foreach capture sequence would have to be fluorescently labeled.

The enzyme-catalyzed reaction has two distinct advantages. First, astable covalent bond forms between the stationary phase and a labeledmonomer, e.g., a Cy5-labeled monomer. This increases the assaysensitivity versus traditional hybridization assays where thefluorescent label is captured by the stationary phase via non-covalentinteractions (duplex formation). Optionally a stringent washing step canbe employed. Second, the polymerase enzyme incorporates thedideoxynucleotide with high fidelity—due to the replication accuracy ofa polymerase, in general only the base that is complementary to thetarget base will react. SBEX is particularly well suited for planarwaveguide technology, benefiting from the increased speed of a washlessassay and increased sensitivity provided by kinetic data.

Using SBEX on the waveguide platform, enables a rapid assays (<5 min)results to be performed that is are able to differentiate between singlenucleotide polymorphic and wild type sequences at temperatures less than50° C.

Fluorescence imaging is sensitive to speed, sensitivity, noise andresolution, and each may be optimized for use in the invention, forexample, speed may be increased to increase assay times. Base extensionmay be detected using a CCD camera, a streak camera,spectrofluorometers, fluorescence scanners, or other known fluorescencedetection devices, which generally comprise four elements, an excitationsource, a fluorophore, a filter to separate emission and excitationphotons, and a detector to register emission photons and produce arecordable output, typically an electrical or photographic output.

Polymerase enzymes useful in the invention are known in the art andinclude, but are not limited to, thermostable polymerases, such as pfu,Taq, Bst, Tfl, Tgo and Tth polymerase, DNA Polymerase I, Klenowfragment, and/or T4 DNA Polymerase. The polymerase may be aDNA-dependent DNA polymerase, a DNA-dependent RNA polymerase, aRNA-dependent RNA polymerase, a RNA-dependent DNA polymerase or amixture thereof, depending on the template, primer and NTP used. Thepolymerase may or may not have proofreading activity (3′ exonucleaseactivity) and/or 5′ exonuclease activity.

The capture molecule and/or the analyte molecule of the invention may beany nucleic acid, including, but not limited to, DNA and/or RNA andmodifications thereto known in the art, and may incorporate5′-O-(1-thio)nucleoside analog triphosphates, .alpha.-thiotriphosphate,7-Deaza-.alpha.-thiotriphosphate, N6-Me-.alpha.-thiotriphosphate,2′-O-Methyl-triphosphates, morpholino, PNA, aminoalkyl analogs, and/orphosphorotioate.

In one embodiment immunoassays can be used with the present method ofusing the scanning sensing system. The optical sensing site of themethod of using the scanning sensing system of the invention can beadapted to support an immunoassay, for example, by including one or moreimmunoassay reagent at or within the optical sensing site. In thisembodiment an interaction between the optical sensing site and a samplebeing tested for a biologically active analyte can include animmunoassay conducted at the optical sensing site. As such, the opticalsensing site interacting with the biologically active analyte caninclude an outcome of an immunoassay. In this manner, presence orabsence of the analyte can be determined. Additionally the amount ofanalyte can be quantified. In one embodiment the immunoassay supportedis a fluorescent assay. It is envisioned that the immunoassay can be acompetitive or non-competitive immunoassay. In one embodiment theimmunoassay supported in an ELISA.

It is envisioned that a variety of instrumentation relating tobiological or environmental sample preparation, handling and analysiscan be used in conjunction with the system and methods described herein.Examples of such instrumentation include but are not limited to a cellsorter, a DNA amplification thermal cycler, or a chromatography machine(e.g., GC or HPLC). Such instrumentation is well known to those skilledin the art. It is envisioned that a robotic interface could be usedbetween the scanning sensing system of the present invention and variousinstrumentation relating to biological or environmental samplepreparation, handling and analysis.

The system and methods described herein may be used in a range ofapplications including biomedical and genetic research as well asclinical diagnostics. Arrays of polymers such as nucleic acids may bescreened for specific binding to a target, such as a complementarynucleotide, for example, in screening studies for determination ofbinding affinity and in diagnostic assays. In one embodiment, sequencingof polynucleotides can be conducted, as disclosed in U.S. Pat. No.5,547,839. The nucleic acid arrays may be used in many otherapplications including detection of genetic diseases such as cysticfibrosis or diagnosis of HIV, as disclosed in U.S. Pat. Nos. 6,027,880and 5,861,242. Genetic mutations may be detected by sequencing byhydridization. In one embodiment, genetic markers may be sequenced andmapped using Type-IIs restriction endonucleases as disclosed in U.S.Pat. No. 5,710,000.

Other applications include chip based genotyping, species identificationand phenotypic characterization, as described in U.S. Pat. No.6,228,575. Still other applications including diagnosing a cancerouscondition or diagnosing viral, bacterial, and other pathological ornonpathological infections, are described in U.S. Pat. No. 5,800,992. Afurther application includes chip based single nucleotide polymorphism(SNP) detection as described in U.S. Pat. No. 6,361,947.

Gene expression may be monitored by hybridization of large numbers ofmRNAs in parallel using high density arrays of nucleic acids in cells,such as in microorganisms such as yeast, as described in Lockhart etal., Nature Biotechnology, 14:1675-1680 (1996). Bacterial transcriptimaging by hybridization of total RNA to nucleic acid arrays may beconducted as described in Saizieu et al., Nature Biotechnology, 16:45-48(1998). Accessing genetic information using high density DNA arrays isfurther described in Chee, Science 274:610-614 (1996).

A non-limiting list of potential application suitable for sensing usingthe systems and methods described herein includes: pathogens detectionand classification; chemical/biological warfare real-time detection;chemical concentration control; dangerous substance (e.g., gas, liquid)detection and alarm; sugar and insulin levels detection in diabeticpatients; pregnancy testing; detection of viral and bacterial infectiousdiseases (e.g. AIDS, Bird Flu, SARS, West Nile virus); environmentalpollution monitoring (e.g., water, air); and quality control in foodprocessing.

The working system described here can also be a sub-system within a muchlarger bio-analysis system. The bio-analysis system could include allthe aspects of sample preparation prior to the optical scanning, thepost processing of data collected in the optical scanning phase andfinally decision making based on these results. Sample preparation mayinclude steps such as: extraction of the sample from the tested subject(human, animal, plant environment etc.); separation of different partsof the sample to achieve higher concentration and purity of themolecules under investigation; sample amplification (e.g. through PCR);attachment of fluorescence tags or markers to different parts of thesample; and spotting of the sample into the sensing chip. The postprocessing of the collected data may include: normalization; backgroundand noise reduction; and statistical analysis such as averaging overrepeated tests or correlation between different tests. The decisionmaking may include: testing against a predefined set of rules andcomparison to information stored in external data-bases.

The applications and uses of the scanning sensing systems describedherein can produce one or more result useful to diagnose a disease stateof an individual, for example, a patient. In one embodiment, a method ofdiagnosing a disease comprises reviewing or analyzing data relating tothe presence and/or the concentration level of a target in a sample. Aconclusion based review or analysis of the data can be provided to apatient, a health care provider or a health care manager. In oneembodiment the conclusion is based on the review or analysis of dataregarding a disease diagnosis. It is envisioned that in anotherembodiment that providing a conclusion to a patient, a health careprovider or a health care manager includes transmission of the data overa network.

Accordingly, business systems and methods using the scanning sensingsystems and methods described herein are provided.

One aspect of the invention is a business method comprising screeningpatient test samples for the presence or absence of a biologicallyactive analyte to produce data regarding the analyte, collecting theanalyte data, providing the analyte data to a patient, a health careprovider or a health care manager for making a conclusion based onreview or analysis of the data regarding a disease diagnosis. In oneembodiment the conclusion is provided to a patient, a health careprovider or a health care manager includes transmission of the data overa network.

Accordingly FIG. 8 is a block diagram showing a representative examplelogic device through which reviewing or analyzing data relating to thepresent invention can be achieved. Such data can be in relation to adisease, disorder or condition in an individual. FIG. 8 shows a computersystem (or digital device) 800 connected to an apparatus 820 for usewith the scanning sensing system 824 to, for example, produce a result.The computer system 800 may be understood as a logical apparatus thatcan read instructions from media 811 and/or network port 805, which canoptionally be connected to server 809 having fixed media 812. The systemshown in FIG. 8 includes CPU 801, disk drives 803, optional inputdevices such as keyboard 815 and/or mouse 816 and optional monitor 807.Data communication can be achieved through the indicated communicationmedium to a server 809 at a local or a remote location. Thecommunication medium can include any means of transmitting and/orreceiving data. For example, the communication medium can be a networkconnection, a wireless connection or an interne connection. Such aconnection can provide for communication over the World Wide Web. It isenvisioned that data relating to the present invention can betransmitted over such networks or connections for reception and/orreview by a party 822. The receiving party 822 can be but is not limitedto a patient, a health care provider or a health care manager.

In one embodiment, a computer-readable medium includes a medium suitablefor transmission of a result of an analysis of an environmental orbiological sample. The medium can include a result regarding a diseasecondition or state of a subject, wherein such a result is derived usingthe methods described herein.

Kits comprising reagents useful for performing the methods describedherein are also provided.

In some embodiments, a kit comprises scanning sensing system asdescribed herein and reagents for detecting a target in the sample. Thekit may optionally contain one or more of the following: one or morefluorescent or luminescent molecular tag, and one or more biologicallyactive analyte including a nucleic acid, protein, microorganism orchemical agent.

The components of a kit can be retained by a housing. Instructions forusing the kit to perform a described method can be provided with thehousing, and can be provided in any fixed medium. The instructions maybe located inside the housing or outside the housing, and may be printedon the interior or exterior of any surface forming the housing thatrenders the instructions legible. A kit may be in multiplex form fordetection of one or more different target biologically active analyteincluding nucleic acid, protein, microorganism, gas, chemical agent orpollutant.

As described herein and shown in an illustrative example in FIG. 9, incertain embodiments a kit 903 can include a housing or container 902 forhousing various components. As shown in FIG. 9, and described herein,the kit 903 can optionally include instructions 901 and reagents 905,for example, DNA hybridization or immunoassay reagents. Otherembodiments of the kit 903 are envisioned wherein the components includevarious additional features described herein.

In one embodiment, a kit for assaying a sample for a target includes ascanning sensor system including a light source, a detector, and asubstrate. The substrate can include a plurality of substantiallyparallel in-coupling waveguides and a plurality of substantiallyparallel out-coupling waveguides as described herein. The system canfurther include a plurality of optical sensing sites. The opticalsensing sites are in optical communication with one or more waveguides.The kit further includes packaging and instructions for use of thesystem.

In one embodiment, the kit includes a scanning sensor system that is aplanar lightwave circuit (PLC).

In general, in another aspect methods of manufacturing a scanningsensing system for assaying a sample for a target are provided. In oneembodiment the system is a PLC.

The starting material or substrate for manufacturing PLC devices is awafer usually made of Silicon (Si) or Silica (SiO2). The most commonwafer diameters in use are 4″, 6″ and 8″. The manufacturing process forPLC devices involves two basic processes namely, deposition and etching.A short description of each of them is given below.

In certain embodiments the methods of manufacturing the systemsdescribed herein can include, but are not limited to laser writing, UVwriting and photonic band-gap waveguide methods. The manufacturingprocess in some embodiments includes one or more steps of deposition,masking and etching.

Deposition:

In the deposition step a layer of well defined material having wellcontrolled thickness is deposited across the entire wafer. The mostcommon material used for waveguide layer deposition is Silica (SiO2)also known as glass. The optical properties of the Silica (mainly itsrefractive index) is controlled by the amount of doping (Ge, P, and Betc.) introduced during the deposition. Other materials such as silicon,glass, epoxy, lithium niobate, indium phosphide and SiON (SiliconOxyNitride) and its derivatives are also used. For the cladding layer,materials can include but are not limited to silicon, silica (SiO2),glass, epoxy, lithium niobate and indium phosphide

The deposition step is done using several technologies such as PECVD(Plasma-Enhanced Chemical Vapor Deposition), LPCVD (Low Pressure CVD),APCVD (Atmospheric pressure CVD), FHD (Flame Hydrolysis Deposition) andothers well known in the art.

FIG. 10A illustrates an exemplary substrate 1004 as a schematicstructure created after two consecutive deposition steps of a cladding1021 layer and a core 1023 layer over a silicon 1020 layer, which can bea wafer. As mentioned above, these two layers differ in the refractionindex which is achieved by using different levels of doping. Typicalthicknesses for the different layers are: Cladding up to about 20 μm andcore up to 6 μm. The thickness of the silicon 1020 wafer can range fromabout 0.5 mm to 1 mm.

Masking:

Following the deposition and before the etching step, the desiredtwo-dimensional structure of the PLC device is transferred to thedeposited wafer by masking the areas not to be etched away. The maskingis done in several steps involving covering the wafer with lightsensitive material, exposing it to light through lithographic masks andremoving the exposed material leaving in place the mask. The result ofsuch steps is shown in FIG. 10B where a mask 1025 is shown on top of thecore 1023 layer of the substrate 1004.

Etching:

In the etching step, material at the un-masked areas is removed from thetop core 1023 layer of the substrate (see FIG. 10C). The etching rate isa known parameter, therefore the etching depth can be controlled bytime. The two most common techniques for etching are wet-etching andReactive-Ion-Etching (RIO). FIG. 10C shows the results of the etchingstep which results in a waveguide 1027.

After the etching step, an over-cladding or top cladding 1029 layer iscreated using a deposition step similar to the one described above. Theresults are shown in FIG. 10D. As shown in FIG. 10D, the resultingwaveguide 1027 can be surrounded by a top cladding 1029 and a cladding1021 over a silicon 1020 layer.

The above steps can be repeated to create several waveguide layers oneon top of the other. In this case, a planarization step may be requiredbetween one waveguide layer and the other. This is done using atechnique known as Chemical Mechanical Planarization (CMP).

When the wafer processing is completed, it can be diced into theindividual chips. An exemplary simplified flow-chart of themanufacturing process is shown in FIG. 11.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A scanning sensing method comprising: coupling aremovable substrate comprising a plurality of in-coupling waveguides, aplurality of out-coupling waveguides and combiners for coupling thein-coupling and out-coupling waveguides, wherein the substrate iscoupled in optical communication with an adapter, a light source, and adetector to provide a scanning sensor system, wherein the adapter iscoupled to the light source and the detector; delivering a samplesuspected of containing a biologically active analyte to be detected toan optical sensing site of the substrate, wherein the optical sensingsite comprises a well; providing a first light wave using a light sourceto one or more of the plurality of in-coupling waveguides of thesubstrate, wherein the in-coupling waveguides are in opticalcommunication with the optical sensing site, wherein the first lightwave is transducable by a sensor associated with the optical sensingsite to a second light wave carried to an out-coupling waveguide; anddetecting the second light wave using the detector, wherein a measurablechange in the first light wave as measured in the second light waveoccurs when the sensor interacts with the biologically active analyte.2. The method of claim 1, wherein scanning sensing further comprisesswitching one or more input light wave from the light source into thesubstrate to produce the first light wave in one or more of thein-coupling waveguides.
 3. The method of claim 1, wherein the lightsource comprises an optical switch for controlled switching of one ormore input light wave, the optical switch can multicast light to aplurality of outputs and into the substrate to controllably produce thefirst light wave in one or more of the in-coupling waveguides.
 4. Themethod of claim 1, wherein the light source comprises an array ofindividually controlled light generators for controlled switching of oneor more input light wave, to controllably produce the first light wavein one or more of the in-coupling waveguides.
 5. The method of claim 1,wherein the incoupling waveguides are in optical communication with aplurality of optical sensing sites and the first wave generates aplurality of second light waves from the plurality of optical sensingsites, the method further comprising simultaneously detecting theplurality of second light waves with the detector wherein the detectorcomprises a photodetector array.
 6. The method of claim 1, wherein, aportion of the sensing sites comprise reference sample material forcalibration and/or normalization.
 7. The method of claim 1, wherein thebiologically active analyte is selected from the group consisting of anucleic acid, a protein, an antigen, an antibody, a lipid, apolysaccharide, a cell, a tissue, a microorganism, a gas, a chemicalagent and a pollutant.
 8. The method of claim 1, wherein thebiologically active analyte is a protein.
 9. The method of claim 1,wherein a SNP is detected in the biologically active analyte.
 10. Themethod of claim 1, wherein a marker for gene expression is detected inthe biologically active analyte.
 11. The method of claim 1, wherein thesensor comprises an immunoassay reagent that supports an immunoassay andwherein the immunoassay reagent of the sensor interacting with thebiologically active analyte comprises an outcome of an immunoassay. 12.The method of claim 11, wherein the immunoassay supported is anenzyme-linked immunosorbent assay (ELISA).
 13. The method of claim 11,wherein the immunoassay supported is a fluorescent immunoassay.
 14. Themethod of claim 1, wherein detecting a measurable change in the firstlightwave as measured in the second lightwave provides a diagnosticresult.
 15. The method of claim 1, further comprising conducting areal-time PCR reaction at the optical sensing site.
 16. A chip fordetecting a biologically active analyte, comprising a substrate whereinthe substrate comprises a plurality of in-coupling waveguides coupled toa plurality of out-coupling waveguides by combiners, where theout-coupling waveguides are in optical communication with a plurality ofsensing sites.
 17. The chip of claim 16, wherein the optical sensingsites comprise a sensor and a sample comprising a biologically activeanalyte, and wherein a measurable change in a first light wave resultswhen the sensor discriminates or interacts with the biologically activeanalyte.
 18. The chip of claim 16 wherein a first light wave in anin-coupling waveguide is transduced by a sensor of an optical sensingsite in optical communication with the out-coupling waveguide resultingin a second light wave in a out-coupling waveguide.
 19. The chip ofclaim 17, wherein the sensor comprises an immunoassay reagent thatsupports an immunoassay.
 20. The chip of claim 19, wherein theimmunoassay supported is an enzyme-linked immunosorbent assay (ELISA).21. The chip of claim 19, wherein the immunoassay supported is afluorescent immunoassay.
 22. The chip of claim 17, wherein the sensor isselected from the group consisting of a fluorescence well, an absorptioncell, an interferometric sensor, a diffractive sensor and surfaceplasmon resonance sensor.
 23. The chip of claim 16, wherein thebiologically active analyte is selected from the group consisting of anucleic acid, a protein, an antigen, an antibody, a lipid, apolysaccharide, a glycoprotein, a cell, a tissue, a microorganism, agas, a chemical agent and a pollutant.
 24. The chip of claim 23, whereinthe nucleic acid is produced via an amplification reaction.
 25. The chipof claim 16, wherein the in-coupling waveguides are single-mode and theout-coupling waveguides are multi-mode.
 26. The chip of claim 16,wherein the in-coupling waveguides and the out-coupling waveguidessupport single-mode in a first vertical dimension and multi-mode in asecond lateral dimension.
 27. The chip of claim 16, wherein thein-coupling waveguides and the out-coupling waveguides are multi-mode.28. The chip of claim 16, wherein the in-coupling waveguides and theout-coupling waveguides are single-mode.
 29. The chip of claim 16,wherein the in-coupling waveguide comprises a plurality of branches fordrawing a fraction of the light from a first light wave traveling in thein-coupling waveguide.
 30. The chip of claim 29, wherein the in-couplingwaveguide branches are in optical communication with the in-couplingwaveguide.
 31. The chip of claim 16, wherein the out-coupling waveguidecomprises a plurality of funnels for collecting light from the sensingsites and coupling it to the out-coupling waveguide.
 32. The chip ofclaim 16, wherein the optical sensing sites comprise wells.
 33. The chipof claim 16, wherein the optical sensing sites comprise the surface ofthe substrate above the out-coupling waveguides.
 34. The chip of claim16, wherein the optical sensing sites comprise biochemical interactionsites.
 35. The chip of claim 16, wherein the optical sensing sitescomprise optical transducers.
 36. The chip of claim 35, wherein theoptical transducers comprise wells comprising fluorescent compounds,wherein light waves guided by the in-coupling waveguides excite thefluorescent compound in the wells in the presence of a biologicallyactive analyte, and the out-coupling waveguides collect and guide lightemitted from the fluorescent compound or the luminescent compounds inthe wells to the detector.
 37. The chip of claim 16, wherein the numberof sensing wells is greater than
 10. 38. The chip of claim 16, whereinthe density of sensing wells is greater than 100 per cm².
 39. The chipof claim 16, wherein the chip is in thermal communication with a thermaltransfer element.
 40. The chip of claim 39, wherein the thermal transferelement is a thermoelectric cooler.
 41. The chip of claim 16, whereineach optical sensing site comprises a thermal transfer element inthermal communication with the optical sensing site.
 42. The chip ofclaim 41, wherein the thermal transfer element comprises a thin-filmheater.
 43. The chip of claim 41, wherein each optical sensing sitefurther comprises a thermistor in thermal communication with the opticalsensing site.
 44. The chip of claim 16, wherein the substrate furthercomprises one or more microchannel and one or more reservoirs in fluidcommunication with one or more optical sensing site.
 45. The chip ofclaim 16, wherein the chip further comprises a fluidics layer coupled tothe substrate and comprising one or more microchannel and one or morereservoirs in fluid communication with one or more optical sensing site.